Method for detecting and identifying microorganism causative of infection

ABSTRACT

Causative microorganisms of infectious diseases are detected and/or identified rapidly and with high sensitivity by taking phagocytes from clinical specimens containing active phagocytes, immobilizing the phagocytes so taken, treating the phagocytes to improve cell membrane permeabilities thereof, further treating the phagocytes to bare DNA in the causative microorganisms which might exist in the phagocytes, and detecting the causative microorganisms with DNA probes which can hybridize with such DNA under stringent conditions.

TECHNICAL FIELD

The present invention relates to an improved method for detecting andidentifying causative microorganisms of infectious diseases. The presentinvention also relates to a kit for detecting and/or identifyingcausative microorganisms of infectious diseases, a method for monitoringgenes from exogenous microorganisms in clinical specimens, and a methodfor determining causative microorganisms of sepsis and those ofbacteriemia.

BACKGROUND ARTS

Although the hemoculture methodologies have popularly been usedconventionally as a mean to verify bacteria in the blood, since thismethodology needs about from 3 to 14 days to culture and isolate thesubjected bacteria and detection rates thereby are as low as about 10%,it was not well contributed in the diagnosis for treating seriousdiseases like sepsis.

The present inventors had invented, to solve such problems, a method fordetecting and identifying exogenous-microorganisms digested withphagocytes comprising a step of detecting genes from suchexogenous-microorganisms in the phagocytes by in situ hybridizationemploying a probe which can specifically hybridize with the genes(Japanese Patent Publication No. 7-40).

The method of Japanese Patent Publication No. 7-40 have been in thelimelight in the field of infectious deseases because, in comparisonwith the conventional hemoculture methodology, the method allowed aboutfour times rapidly detection of the subjected bacteria in bloods frompatients who are under the suspicion about sepsis, and detection resultswere appeared within 24 hours.

Objects of the present inventions is an improvement of detection effectsand of detection sensitivity to be offered by the method according toJapanese Patent Publication No. 7-40 for detecting and/or identifyingcausative microorganisms of infectious diseases by taking phagocytesfrom the clinical specimens containing active phagocytes, immobilizingthe phagocytes so taken, treating the phagocytes to improve cellmembrane permeabilities thereof, further treating the phagocytes to bareDNA in the causative microorganisms which might be existed in thephagocytes, in situ hybridizing DNA so bared with detective DNA probe(s)which can hybridize with such bared DNA under stringent conditions, anddetecting and/or identifying the causative microorganisms based onsignals so detected.

DISCLOSURE OF INVENTION

The present invention has been completed in view of the problemsaforenoted and the merits thereof are as follows.

A method for detecting and/or identifying causative microorganisms ofinfectious diseases by taking phagocytes from the clinical specimenscontaining active phagocytes, immobilizing the phagocytes so taken,treating the phagocytes to improve cell membrane permeabilities thereof,further treating the phagocytes to bare DNA in the causativemicroorganisms which might be existed in the phagocytes, in situhybridizing DNA so bared with detective DNA probe(s) which can hybridizewith such bared DNA under stringent conditions, and detecting and/oridentifying the causative microorganisms based on signals so detected,the method comprises at least one condition(s) to be selected from thefollowing conditions (1)-(8) of;

(1) Cell density (X cells/ml) of the phagocytes to be immobilized is5×10⁶ cells/ml<X cells/ml<1×10⁸ cells/ml,

(2) Lysostaphin is applied into the step to bare DNA in the titer offrom 1 Unit/ml to 1,000 Units/ml,

(3) Lysozyme is applied into the step to bare DNA in the titer of from1,000 Units/ml to 1,000,000 Units/ml,

(4) N-acetylmuramidase is applied into the step to bare DNA in the titerof from 10 Units/ml to 10,000 Units/ml,

(5) Zymolyase is applied into the step to bare DNA in the titer of from50 Units/ml to 500 Units/ml,

(6) Surfactant is applied into the step of in situ hybridization,

(7) Such DNA probe(s) is/are one or more DNA probe(s) to be determinedwith their chain length of from 350 bases to 600 bases, and

(8) Concentration of such DNA probe(s) is from 0.1 ng/μl to 2.2 ng/μl.

The step to bare DNA employs preferably one or more enzyme(s) selectedfrom Lysostaphin in the titer of from about 10 to about 100 Units/ml,Lysozyme in the titer of from about 10,000 to about 100,000 Units/ml,N-acetylmuramidase in the titer of from about 100 to about 1,000Units/ml and Zymolyase in the titer of from about 100 to about 500Units/ml.

The step to bare DNA preferably employs enzyme(s), and the enzyme(s)is/are subjected to a reaction to be performed under the temperature offrom about 26° C. to about 59° C. for from about 15 to about 120minutes.

The step to bare DNA further employs preferably substance(s), inparticular, phenylmethylsulfonyl fluoride to keep a form of thephagocytes under the concentration of preferably from about 10 μmol/l toabout 10 mmol/l.

As the substance to keep a form of the phagocytes, substance dissolvedinto dimethylsulfoxide is preferable. The substance dissolved indimethylsulfoxide is employed as that to keep a form of the phagocytes,then, concentration of the dimethylsulfoxide in a solution to be used inthe step to bare DNA is adjusted to less than 5%.

The in situ hybridization step is performed by hybridizing DNA with DNAprobe(s) under the presence of surfactant(s), in particular, the anionicsurfactant, preferably, sodium dodecyl sulfate (SDS).

Hybridization reaction in the in situ hybridization step is performedunder the temperature of from about 20° C. to about 50° C. and time offrom about 30 to about 900 minutes.

The method further comprises, prior to the immobilization step, a stepto mount the phagocytes so taken onto the solid support and a slidecoated with 3-aminopropyl triethoxysilane is employed as such solidsupport.

Pigment(s) is/are also employed at the signal detection to distinguishcontrast between signals and cells. Preferably, blood is employed as theclinical specimen.

The present invention further provides a kit for detecting and/oridentifying causative microorganisms of infectious diseases by takingphagocytes from the clinical specimens containing active phagocytes,immobilizing the phagocytes so taken, treating the phagocytes to improvecell membrane permeabilities thereof, further treating the phagocytes tobare DNA in the causative microorganisms which might be existed in thephagocytes, in situ hybridizing DNA so bared with detective DNA probe(s)which can hybridize with such bared DNA under stringent conditions, anddetecting and/or identifying the causative microorganisms based onsignals so detected, the kit comprises the following elements (1)-(2)of;

(1) at least one enzyme(s) to be employed in the step to bare DNA whichis/are selected from Lysostaphin, Lysozyme, N-acetylmuramidase andZymolyase; and

(2) at least one detective DNA probe(s).

Then, the present invention further provides a method for monitoring agene of exogenous microorganisms digested with the phagocytes in theclinical specimens containing active phagocytes comprising the step ofdetecting the gene with in situ hybridization method employed in theforegoing method, wherein the gene of exogenous microorganisms in theclinical specimens is monitored.

The present invention further provides a method for diagnosing sepsis orbacteriemia comprising the step of identifying a gene of candidatecausative microorganisms with in situ hybridization method employed inthe foregoing method, wherein the causative microorganisms for sepsis orbacteriemia are determined based on the identification results.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view of illustrating results of in situ hybridizationperformed under (a) the absence of surfactant (SDS) and (b) the presenceof surfactant (SDS).

FIG. 2 is a view of illustrating a manner of leukocytes immobilized withthe various cell density.

FIG. 3 is a view of illustrating a time-coursely change on lytic enzymeactivity against (a) Staphylococcus aureus and Staphylococcusepidermidis, (b) Pseudomonas aeruginosa and Escherichia coli, and (c)Enterococcus faecalis.

FIG. 4 is a view of illustrating concentration-dependency effects byaddition of DMSO against lytic activity to be offered by (a) 300Units/ml of N-acetylmuramidase, (b) 10,000 Units/ml of Lysozyme, and (c)50 Units/ml of Lysostaphin.

FIG. 5 is a view of illustrating results on addition of (a) protease 0.2Units/ml only, (b) PMSF 1 μmol/ml, (c) PMSF 10 μmol/ml, (d) PMSF 0.1mmol/ml, and (e) PMSF 1 mmol/ml to study effects of PMSF to be used tosuppress the function of protease which changes a morphological form ofleukocyte.

FIG. 6 is a view of illustrating that, in the phagocytosis samplesprepared according to the present invention, phagocytes digestedbacteria and morphological forms of such digested bacteria were changed.

FIG. 7 is a view of illustrating effects of enzyme-treatment in thephagocytosis samples and manner of the phagocytosis samples containing(a) Staphylococcus aureus prior to the treatment, (b) Enterococcusfaecalis prior to the treatment, (c) Staphylococcus aureus of (a) withthe treatment, and (d) Enterococcus faecalis of (b) with the treatment.

FIG. 8 is a schematic view of the slide on which the phagocytosissamples were smeared to study optimum probe concentration under the insitu hybridization.

FIG. 9 is a schematic view of the slide on which the phagocytosissamples were smeared to study optimum temperature under the in situhybridization.

FIG. 10 is a view of illustrating the signals appeared in the results ofSouthern Blot (the upper row) and Electrophoresis (the lower row)together with the length of the labelled detective probes prepared byputting digoxigenin-labels on (a) SA probes and (b) PA probes.

FIG. 11 is a view of illustrating the signals detected by in situhybridization of the digested Escherichia coli with detective probes of(a) EC-24, (b) EC-34, (c) EC-39 and (d) the mixed probes (MIX) of theforegoing probes (a)-(c).

BEST MODE FOR CARRYING OUT THE INVENTION

Any clinical specimen containing active phagocytes is applicable as aspecimen to be employed in the present embodiment and may includesfluidus like blood, histofluid, lymph, neurolymph, pus, pituita, nasalmucus or sputum. Then, active phagocytes are contained in urea, ascitesor dialysate in the disorder like diabetes, nephropathia or hepatopathyand are also remained in the lotion used to wash rhinonem, bronchus,skin, various organs or bones, they can therefore also be employed asspecimen of the present invention.

In addition thereto, tissues taken from skin, lung, kidney, mucosa orthe like are also employed as the clinical specimen in the presentinvention. This is because that macrophage, which is one of phagocytes,has various forms including monocytes, alveolar macrophage, celiacmacrophage, fixed macrophage, free macrophage, Hansemann's macrophage,inflammatory macrophage, liver Kupffer cells and brain microglia cells,tissues containing those can therefore also be employed, besides blood,as the clinical specimen of the present invention. For example,causative microorganisms of nephritis can be detected and identified bycollecting kidney tissues through biopsy from patients who are under thesuspicion about nephritis, taking phagocytes in the tissues throughdigestion thereof with enzymes like trypsin to exfoliate cells, andutilizing the phagocytes so taken.

The term ‘phagocytes’ used herein is directed to any cell which canincorporate into itself foreign objects like exogenous microorganismsand may includes, for example, macrophage, monocytes, neutrophil andeosinophil. Phagocytes line like U937 Cell, HL60 Cell or the like isalso available. Exogenous microorganisms which may cause infectiousdiseases are microorganisms to be digested with phagocytes and mayincludes, for example, bacteria, mycete, virus, protozoon, parasite orthe like. Bacteria may include, for example, Staphylococcus,Pseudomonas, Enterococcus, Colibacillus, Streptococcus, Pneumococcus,Tubercle bacillus, Helicobacter pylori, Listeria, Yersinia, Brucellar orthe like. Mycete may include, for example, Candida, Aspergillus,Actinomyces, Coccidioides, Blastomyces or the like. Virus may includeInfluenza virus, Poliovirus, Herpes virus, Hepatitis virus and AIDSvirus. Protozoon may include, for example, Karyamoebina falcata,Trichomonas vaginalis, Malaria, Toxoplasma or the like. Parasite mayinclude, for example, Trypanosoma or the like. In particular, thecausative microorganisms of sepsis or bacteriemia may include, forexample, Gram-Positive Bacteria of Staphylococcus genus (Staphylococcusaureus, Staphylococcus epidermidis) and Enterococcus genus (Enterococcusfaecalis, Enterococcus faecium, Streptococcus pneumoniae, Streptococcuspyogenes, Streptococcus agalactiae), Gram-Negative Bacteria likeColibacillus-related Enterobacteriaceae Family of Escherichia coli,Enterobacter cloacae, Klebsiella pneumoniae (Klebsiella oxytoca,Serratia marcesens, Proteus vulgaris, Citrobacter freundii), aerophilicrod of Pseudomonas genus (Pseudomonas aeruginosa), anaerobe ofClostridium genus (Clostridium perfringens), Bacteroides genus(Bacteroides fragilis) or the like. Also, Acinetobacter calcoaceticus,Aeromonas hydrophilia, Flavobacterium meningosepticum, Bacillus cereuscan rarely be fallen within the causative microorganisms.

Phagocytes (Leukocytes) fractions can be taken from the clinicalspecimen according to the conventional method. For example, about 5 ml(or 10 ml with a few leukocyte) of heparinized venous bloods wereobtained and the bloods were mixed with the blood components separativereagent (adjusted with sterilized-purified water to be 25 ml as theirfinal volume containing 225 mg of Sodium Chloride and 1.5 g of Dextran(Molecular Weight: 200,000-300,000)) in the ratio of 4:1. Leukocytefractions (the upper layer) were then obtained by leaving them at fromabout 10 to about 40° C. for from about 15 minutes to about 120 minutes,preferably, about 37° C. for about 30 minutes. Leukocytes were appearedby centrifuging the leukocyte fractions so taken in from about 100×g toabout 500×g, at from 0° C. to about 20° C. for about from 3 minutes toabout 60 minutes, preferably, in from about 140×g to about 180×g, atabout 4° C. for about 10 minutes. Hemolysis is preferable iferythroblasts are entered at this step. Pelletized leukocytes soobtained were, for example, suspended with 1 ml of sterilized-purifiedwater and the suspension were immediately put into an isotonic state byadding thereto excessive amounts of PBS (prepared by dilutingtwenty-fold with sterilized-purified water the raw solution (PBS RawSolution; hereinafter simply referred to as ‘PBS Raw Solution’) whichhave been adjusted with sterilized-purified water to be 120 ml as theirfinal volume containing 18.24 g of Sodium Chloride, 6.012 g of SodiumMonohydrogen Phosphate Didecahydrate and 1.123 g of Sodium DihydrogenPhosphate Dihydrate), and such suspension were re-centrifuged in fromabout 140×g to about 180×g, at 4° C. and for about 10 minutes.Otherwise, such digested phagocytes can be adhered to the slides to benoted later through their native adhesionability without anycentrifugation aforenoted.

Methodology to fix the leukocytes may include, for example, Carnoy sfixation. In particular, leukocytes are mounted onto a support (asupportive medium) which can hold the leukocytes and are immersed inCarnoy s fixative solution (prepared by mixing Ethanol, Chloroform andAcetic Acid in the volume ratio of 6:3:1) for 20 minutes and wereimmersed in from about 50% to about 90%, preferably, about 75% Ethanolsolution for five minutes. Finally, they were completely air-dried.

Insoluble materials are preferable for the support and may include, forexample, glass, metal, synthesized resin (e.g., polystyrene,polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylicester, nylon, polyacetal and fluoric resin) and polysaccharide (e.g.,cellulose and agarose).

A form of the insoluble support can be changed optionally and mayinclude, for example, that of plate, tray, ball, fiber, rod, disk,container, cell or tube.

Particularly preferred support in the present embodiment is to emplyslides. Such slide may include, for example, the slide (PRODUCTS ID.S311BL) of JAPAN AR BROWN CO., LTD. This slide (PRODUCTS ID. S311BL) has14 circular wells of 5 mm diameter. In order to improve anadhesionability of the subjected cells at the actual use, APS coatedslides to be made by coating 3-aminopropyltriethoxysilane (APS, SIGMA)onto the slides are recommended. The other slides to be made by coatingpoly-L-lysine or gelatin are also available.

APS coated slides are prepared by putting the slides (PRODUCTS ID.S311BL) onto a holder, immersing them into the diluted neutral detergentfor 30 minutes or more, removing well the detergent with tap water, thenwashing the slides with purified water, and drying well the same underthe higher temperature (100° C. or more) followed by leaving it underthe room temperature. Thereafter, these slides are immersed into acetonecontaining 2% APS for one minute and are immediately rinsed gently withacetone then with sterilized-purified water. These slides were thenair-dried. These slides are re-immersed into acetone containing fromabout 1° C. to about 10% APS for one minute, are immediately rinsedgently with acetone then sterilized-purified water, and are air-dried.Finally, these slides are dried under the temperature of from about 20°C. to about 60° C., preferably of 42° C. to realize the APS coatedslides.

Leukocytes are mounted onto the APS coated slides preferably by smearingthe leukocytes to realize the extended mono-layer thereof and drying thesame. Cell population (X cells/ml) of the immobilized phagocytes shouldbe adjusted to that of about 5×10⁶ cells/ml<X cells/ml<about 1×10⁸cells/ml, preferably, about 1×10⁷ cells/ml≦X cells/ml≦about 5×10⁷cells/ml.

Then, according to change on the phagocytes population per 1 ml,leukocyte population to be immobilized in the single well of the APScoated slide (y cells/well (diameter: 5 mm)) are adjusted that of about2.5×10⁴ cells/well<y cells/well<about 5×10⁵ cells/well, preferably,about 5×10⁴ cells/well≦y cells/well≦about 2.5×10⁵ cells/well. Inparticular, pelletized leukocytes are prepared by centrifuging leukocytefractions in from about 140×g to about 180×g, at 4° C. and for 10minutes, adding small amount of PBS to the pelletized leukocytes,suspending the same, and counting the population with a hemacytometer.Leukocytes were duly mounted on the APS coated slides by smearing 5 μlof the leukocyte suspension into each well of the slides adjusted withPBS to be cell population of from about 5×10⁴ cells/well to about2.5×10⁵ cells/well, then extending mono-layer of the leukocytes, andcompletely air-drying the same.

In order to accelerate permeability of the phagocyte membrane, they wereimmersed in PBS for from about 3 minutes to about 30 minutes, then inthe solution prepared by diluting from about 2-fold to about 50-fold apretreatment reagent (prepared by mixing 1.25 g of Saponin, 1.25 mlt-octylphenoxy-polyethoxyethanol (specific gravity of 1.068-1.075 (20/4°C.), pH (5 w/v %) 5.5-7.5) and 25 ml PBS Raw Solution, and adjustingwith sterilized-purified water to be 50 ml as their final volume), andwere applied to a centrifuge for from about 3 minutes to about 30minutes.

In order to bare DNA in the causative microorganisms, an enzyme solutionwas prepared by adding, per single slide, 1 ml of a reagent solvent(prepared by diluting about 100-fold, with PBS, Dimethyl Sulfoxide(DMSO) containing 0.1 mol/l Phenylmethyl Sulfonylfluoride (PMSF)) to anenzyme reagent (N-acetylmuramidase, Lysozyme and/or Lysostaphin), then 1ml of which were dropped under the temperature of from about 20° C. toabout 60° C., preferably, from about 37° C. to about 42° C. in the wetchamber onto the area where the leukocytes were smeared and were leftfor from about 10 minutes to about 60 minutes. Then, they were immersedin PBS containing 0.2 mol/l Hydrochloric Acid (prepared by addinghydrochloric acid to PBS Raw Solution, diluting 20-fold the same withsterilized-purified water and adjusting final concentration ofHydrochloric Acid to 0.2 mol/l), and were applied to a centrifuge for3-30 minutes to accelerate their permeability. 5% or more of DMSOconcentration may lower activities of Lysozyme and Lysostaphin, DMSOconcentration of less than 5% is therefore preferable. Besides PMSF, theknown protease inhibitors like tosyllysinechlromethylketone (TLCK) and acombination thereof are also applicable to keep the form of phagocytes.Solvents like DMSO can be changed optionally to employ such knownprotease inhibitors.

With regard to the preferable titer range on each enzyme employed in theenzyme reagent, although Lysostaphin offer the substantial effect at thetiter of 1 Unit/ml in the lysis of Staphylococcus aureus, that of 10Units/ml or more was necessary in the lysis of Staphylococcusepidermidis. Optimum titer on Lysostaphin should therefore be adjustedto from about 1 Unit/ml to about 1,000 Units/ml, preferably, from about10 Units/ml to about 100 Units/ml. When the titer of Lysozyme was about10,000 Units/ml, there was no lysis on Enterococcus faecalis withN-acetylmuramidase of about 10 Units/ml or less titer. Also, when thetiter of N-acetylmuramidase was about 100 Units/ml, there was no lysiswith Lysozyme of about 1,000 Units/ml or less titer. Accordingly,optimum titer on N-acetylmuramidase should be adjusted to from about 10Units/ml to about 10,000 Units/ml, preferably, from about 100 Units/mlto about 1,000 Units/ml, while that on Lysozyme acetylmuramidase shouldbe adjusted to from about 1,000 Units/ml to about 1,000,000 Units/ml,preferably, from about 10,000 Units/ml to about 100,000 Units/ml. Whenthe causative microorganisms are mycete like Candida albicans, titer onZymolase should be adjusted to from about 50 Units/ml to about 500Units/ml, preferably, from about 100 Units/ml to about 500 Units/ml. Inparticular, PMSF or the known protease inhibitors are useful incombination with Zymolase.

Enzyme(s) could be selected based on the components difference betweenGram-Positive Bacteria and Gram-Negative Bacteria, namely, thedifference of peptideglycan or of lipopolysaccharides. Two kinds or moreof enzymes are particularly preferable to effectively lyse bothGram-Positive Bacteria and Gram-Negative Bacteria. It was demonstratedin the present invention that the lysis activities offered by the mixedenzymes of Lysozyme, Lysostaphin and N-acetylmuramidase were enhanced incomparison with those by the single enzyme.

Treatment temperature with enzymes on Staphylococcus aureus shouldpreferably be adjusted to from about 4° C. to about 60° C., then that onStaphylococcus epidermidis should be adjusted to about 25° C. or more,preferably, to about 37° C. or more, and that on Enterococcus faecalisshould be adjusted to from about 25° C. to less than about 60° C.,preferably, from about 37° C. to about 42° C. Accordingly, it is mostpreferable to designate the temperature range of from about 37° C. toabout 42° C. as the optimum treatment temperature. Further, the criticaltemperature range to be shared with the three bacterial species isexpected to the range of from about 26° C. to about 59° C.

Then, treatment time with enzymes on any digested sample fromStaphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalisis 20 minutes or more (both at zero minute and 10 minutes were notappropriate), then, since there was no leukocyte in the bacteria, thetreatment time should be adjusted to at least about 15 minutes or more,preferably, to about 20 minutes or more, and be designated the timerange of from about 30 minutes to about 60 minutes as the optimumtreatment time. Treatment time of from about 15 minutes to about 120minutes may also be applicable.

Then, N-acetylmuramidase is an enzyme which lower an absorbance at 600nm under the reaction at 37° C. for 5 minutes of N-acetylmuramidase withheat-treated dry-powder of Enterococcus faecalis in 5 mmol/l Tris-HClBuffer Solution (pH 6.0) containing 2 mmol/l magnesium chloride. Then,when 1 Unit of enzyme activity is defined as an activity to lyse one ugof heat-treated dry-powder of Streptococcus salivarius (IFO 3350) in oneminute which was determined thereon at 37° C. and pH 7.0, the enzyme of2,000 Units/mg or more is preferable.

Lysozyme is an enzyme which lower an absorbance at 600 nm under thereaction of Micrococcus luteus with Lysozyme in PBS at 37° C. for 5minutes. Then, when 1 Unit of enzyme activity is defined as an activityto lower 0.001 of an absorbance at 540 nm in one minute which wasdetermined on Micrococcus luteus at 35° C. and pH 6.2, an enzyme of50,000 Units/mg or more is preferable.

Lysostaphin is an enzyme which lower an absorbance at 600 nm under thereaction of Staphylococcus epidermidis with Lysostaphin in PBS at 37° C.for 5 minutes. When 1 Unit of enzyme activity is defined as an activityto lower an absorbance at 620 nm of 0.240 to 0.125 in 10 minutes whichwas determined on Staphylococcus aureus at 37° C. and pH 7.5, an enzymeof 500 Units/mg or more is preferable.

Zymolase (Products Name: Zymolyase (SEIKAGAKU CORPORATION)) is an enzymetaken from the liquid culture medium of Arthrobacter lutesul and havestrong degradation activities on cell walls of the active yeast cells.Essential enzymes contained in Zymolase and involved with cell walldegradation is β-1,3-glucan lanimaripentaohydrolase which acts onglucose polymer with β-1,3-bonds and produces laminaripentaose as a mainproduct. Zymolyase-100T is purified in ammonium sulfate fractionation,then in affinity chromatography (Kitamura, K. et al., J. Ferment.Technol., 60, 257, 1982) and have activity of 100,000 Units/g. However,it is well known that activities of the subjected enzyme are changedaccording to the kinds of yeast to be substrates, culture condition andgrowth phase thereof (Kitamura, K. et al., J. Gen. Appl. Microbiol., 20,323, 1974; Kitamura, K. et al., Agric. Biol. Chem., 45, 1761, 1981;Kitamura, K. et al., Agric. Biol. Chem., 46, 553, 1982). Zymolyase-100Tcontains about 1.0×10⁷ Units/g of β-1,3-glucanase, about 1.7×10⁴ Units/gof protease and about 6.0×10⁴ Units/g of mannase, but does not containany DNAse and RNAse (Kitamura, K. et al.; J. Gen. Appl. Micro-biol., 18,57, 1972). Then, the optimum pH of Zymolyase is from about 5.5 to about8.5, preferably, from about 6.5 to about 7.5, while the optimumtemperature thereof is from about 25° C. to about 55° C., preferably,from about 35° C. to about 45%. Further, lytic spectrum (genus) to yeast(cells in logarithmic growth phase) may includes Ashbya, Candida,Debaryomyces, Eremothecium, Endomyces, Hansenula, Hanseniaspora,Kloekera, Kluyveromyces, Lipomyces, Helschkowia, Pichia, Pullularia,Torulopsis, Saccharomyces, Saccharomycopsis, Saccharomycodes,Schwanniomyces or the like.

In particular, Candida genus may include Candida albicans, Candidatropicalis, Candida parasilosis, Candida galacta, Candidaguilliermondii, Candida krusei, Cryptococcus neoformans. SH compounds,for example, cysteine, 2-mercapto-ethanol, dithiothreitol can beemployed as an activator of these enzymes.

Bacteria belonged to these genus may also be employed in the presentinvention. 1 Unit of the enzyme activity is defined as an activity tolower about 30% an absorbance at 800 nm of the reaction solution(prepared by adjusting with 1 ml of sterilized-purified water to be 10ml as their final volume containing 1 ml enzyme solution of 0.05˜0.1mg/ml, 3 ml of Brewer's Yeast Suspension (2 mg dry-weight/ml) as asubstrate, and 5 ml of M/15 Phosphoric Acid Buffer Solution (pH 7.5)) intwo hours which was determined on Brewer's Yeast Suspension as asubstrate at about 25° C. Zymolyase-100T has an activity of 100,000Units/g.

With regard to the concentration of PMSF (to be added to keep a form ofthe leukocytes by protecting them from protease) to be used as a reagentsolvent, since 10 μmol/l or more of PMSF concentration was effective anda form of the leukocyte was completely kept at the PMSF concentration of0.1 mmol/l or more, PMSF concentration should be adjusted to the rangeof from about 10 μmol/l to about 10 mmol/l, preferably that of fromabout 0.1 mmol/l to about 1 mmol/l. Similarly, DMSO concentration shouldalso be adjusted to less than 5%, preferably 2% or less, and morepreferably about 1%. Accordingly, the reagent solvent should preferablybe prepared by dulting 100 to 1,000-fold, with PBS, Dimethyl Sulfoxide(DMSO) containing 0.1 mol/l Phenylmethyl Sulfonylfluoride (PMSF).

Acetylation of cell membrane proteins may further be performed after thestep to bare DNA in the causative microorganisms. In particular, slidesare immersed in the acetylation reagent prepared by adding AceticAnhydrate to the acetylation raw reagent (adjusted with quantumsufficient of sterilized-purified water to be 50 ml as their totalvolume containing 7.46 g of Triethanolamine and quantum sufficient ofHydrochloric Acid), diluting from about two-fold to about 50-fold,preferably about 10-fold with sterilized-purified water, and adjustingfinal concentration of Acetic Anhydride to from 0.1% to 3.0%, preferably0.8%, and are applied to a centrifuge for from 5 to 30 minutes. Afterthen, they are completely air-dried by immersing them successively into75%, 85% and 98% Ethanol for from two to five minutes respectively.

Alkalization of DNA in the causative microorganisms may further beperformed to reshape it into the single strand DNA after the step toacetylate the cell membrane proteins. In particular, slides are immersedfor from about two minutes to about five minutes in PBS containing fromabout 10 mmol/l to about 300 mmol/l Sodium Hydroxide, preferably, about70 mmol/l Sodium Hydroxide (prepared by adding Sodium Hydroxide to PBSRaw Solution, diluting 20-fold with sterilized-purified water, andadjusting final concentration of Sodium Hydroxide to 70 mmol/l). Afterthen, they were completely air-dried by immersing them successively into75%, 85% and 98% Ethanol for from two to five minutes respectively.

In order to perform in situ hybridization with detective DNA probe whichcan hybridize under the stringent condition to the naked DNA from thecausative microorganisms, probe solution (solution containing thedetective DNA probe prepared with probe dilution) is applied to the areato be smeared, and is left for from about one hour to about three hours,preferably, for about two hours in the wet chamber of from about 25° C.to about 50%, preferably, from about 37° C. to about 42%.

Then, three stained bottles containing the hybridization detergent(prepared by mixing Hybridization Raw Solution (prepared withsterilized-purified water to be 75 ml as their total volume containing13.15 g of Sodium Chloride, 6.615 g of Trisodium Citrate Dihydrate) inthe ratio of Hybridization Raw Solution:sterilized-purifiedwater:Formamide=5:45:50) are provided and they are successively immersedtherein at from about 35 to about 45%, preferably, at about 42° C. for10 minutes respectively. They are then immersed in PBS and are appliedsuccessively to centrifugation on a centrifuge for from about five toabout 30 minutes. In particular, the diluted probe solution contains 600μl of Salmon Sperm DNA, 50 μl of 100×Denhurt's Solution, 500 μl ofHybridization Raw Solution, 2250 μl of Formamide and 1000 μl of 50%Dextran Sulfate. Probe solution contains preferably 15 ng of eachdetective DNA probes and may be adjusted with the probe dilution to be50 μl as their total volume.

Probe concentration of SA, SE, PA, EF, EK is adjusted to from about 0.6ng/μl to about 1.8 ng/μl, preferably to from about 0.6 ng/μl to about1.2 ng/μl. Since result at 0.06 ng/μl was not acceptable while result at0.6 ng/μl was acceptable, it is preferably to adjust the concentrationto at least 0.1 ng/μl or more. Then, since result at 2.4 ng/μl was notacceptable while result at 1.8 ng/μl was acceptable, it is preferably toadjust the concentration to 2.2 ng/μl or less. Further, the optimumconcentration of the positive control and the negative control areadjusted to concentration of from 0.4 ng/μl to 2.0 ng/μl and from 0.6ng/μl to 2.0 ng/μl respectively, preferably, that of from 0.6 ng/μl to1.0 ng/μl for the both control.

Time to perform hybridization is at least 30 minutes or more, preferably60 minutes or more, and more preferably 90 minutes or more. Time of fromabout 120 minutes to about 900 minutes may be designated as optimumhybridization time.

It is preferably to use surfactants like sodium dodecyl sulfate (SDS) inthe step of in situ hybridization because it may allow enhancement ofthe detection sensitivity. SDS concentration is preferably 1% or less,more preferably from about 0.1% to about 0.5%, more further preferablyabout 0.25%. SDS is added to the solution to be employed at thehybridization, otherwise, it may be added in advance to the probediluent or the probe solution.

It is recommended to employ, as the detective DNA probes, one or morekind(s) of DNA probe(s) having from about 350 to about 600 base length,preferably from about 350 to about 550 base length, because the probesare thereby smoothly incorporated into the phagocytes and they mayeasily and exactly be contacted with genes of the exogenousmicroorganisms wherein such probes are incorporated. It is not necessaryto fall base length (number of bases) of the subjected within the baselength range aforenoted, but is simply recommended to employ probeshaving base length distribution to be at least partially overlapped withthe base length range aforenoted. These probes are made of single orseveral (one or more) kind(s) of probes. One or more probe(s) may beplural kinds of probes to be hybridized to the single bacterial speciesor may be plural kinds of probes to be hybridized to the pluralbacterial species one to one, but any restriction would not be imposedas far as one or more kind(s) of probes is/are used.

These probes have preferably DNA fragments containing a sequence not tobe hybridized anyway to phagocytes themselves and would notcross-hybridize at all to any gene from the unrelevant bacteria species.Specific probes would be prepared in a short time, for example, with asubtraction method. These probes may be prepared and labelled throughthe conventional nick translation methodology using non-radioisotopiclabelling substances like fluorescein isothiocyanate (FITC), biotin,digoxigenin (digoxigenin(DIG)-11-dUTP) or the like. Strand length of theprobes can be adjusted most effectively by changing the amount ratio ofDNAseI and DNA polymerase I respectively to be added at the nicktranslation reaction. For example, in order to effectively label 2 μg ofDNA probe (SA-24) and adjust strand length thereof (into base length offrom about 350 to about 600) which allow the probes to be effectively insitu hybridized to the exogenous microorganisms DNA, in the reactionarysolution of 100 μl total volume, with regard to 2 μl of 10 U/μl DNApolymerase I, 6 μl of DNAseI is presented in the 100 μl total volume asan activity of from about 10 mU to about 350 mU, preferably from about25 mU to about 200 mU, and more preferably from about 50 mU to about 150mU. As far as volume ratio in the essential optimum reaction conditionsaforenoted is kept at the certain level, volume of each enzyme and totalvolume of the reactionary solution can be changed optionally. In theother words, volume of DNAseI may be adjusted, with regard to 20 U ofDNA polymerase I in the total volume of 100 μl, to from about 10 mU toabout 350 mU, preferably from about 25 mU to about 200 mU, and morepreferably from about 50 mU to about 150 mU. Turning to the otheraspect, it is recommended to perform nick translation reaction byadjusting volume of DNAseI, with regard to 1 Unit of DNA polymerase I,to from about 0.5/1,000 U to about 17.5/1,000 U, preferably from about1.25/1,000 U to about 10/1,000 U, and more preferably from about2.5/1,000 U to about 7.5/1,000 U. Then, with regard to 1 μg of DNA,volume of DNA polymerase I is adjusted to about 10 U, while that ofDNAseI is adjusted to from about 5 mU to about 175 mU, preferably fromabout 12.5 mU to about 100 mU, and more preferably from about 25 mU toabout 75 mU. With regard to the other probes, an amount of DNA as wellas optimum reaction conditions on DNA polymerase and DNAse I aredetermined by referring to the optimum reaction conditions aforenoted,then the probe length (the length of from about 350 bases to about 600bases) is also determined in which the length may allow the probes to beeffectively labelled and may allow the probes to be effectively in situhybridized to the exogenous microorganisms DNA.

Stringent condition to be employed at the in situ hybridization is acondition, for example, to incubate under the presence of formamide offrom about 30% to about 60%, preferably at about 50%, at from about 30°C. to about 50° C., preferably from about 38° C. to about 42° C., andrinse successively.

After in situ hybridization, blocking operation may also be performed.In particular, 1 ml of Blocking reagent (prepared withsterilized-purified water to be 10 ml as their total volume containing 2ml of Rabbit Normal Serum and 0.5 ml of PBS Raw Solution) per singleslide is dropped onto the smeared area thereof and the slides were leftfor from 15 to 60 minutes. Then, Blocking reagent is removed.

In order to detect the signals resulted from hybridization with thebacterial gene (genome DNA or RNA), any coloration reaction utilizingthe conventional antigen-antibody reaction or the like may be employed.Namely, the samples so hybridized are fully washed, then are subjectedto Blocking, and are treated with complexes like anti-FITC antibody,anti-digoxigenin antibody, for example, alkaline phosphatase complexes,followed by evaluation on the hybridization results through signals tobe expressed by the chromogenic substrates in the complexes. Forexample, when a probe is labelled with digoxigenin 11-dUTP as notedabove, anti-digoxigenin alkaline phosphatase complexes will be employed,and the probe may be detected with a substrate (Nitro Blue Tetrazolium,5-Bromo-4-Chloro-3-Indolyl Phosphate or the like) to be usually employedon the alkaline phosphatase. Then, the smear samples, which are washedafter the coloration reaction, are subjected to counterstain withNaphthol Black, Fast Green (20 mg/50 ml, Wako Chemicals) or the like,and in-cell signals are detected by an optical microscopy.

Particularly, in order to take signals in hybridization by employing,for example, DNA probes labelled with digoxigenin as detective DNAprobes, labelled antibody solution is prepared by diluting from 10 to200-fold, preferably 50-fold the labelled antibody (prepared with 12.6μl of Buffer A (prepared with quantum sufficient of sterilized-purifiedwater to be 100 ml as their total volume containing 746 mg ofTriethanolamine, 17.5 mg of Sodium Chloride, 20.3 mg of MagnesiumChloride Hexahydrate, 1.36 mg of Zinc Chloride, 1000 mg of Bovine SerumAlbumin and quantum sufficient of Hydrochloric Acid) to be 14 μl astheir total volume containing 1.05 Unit of alkaline-phosphatase-labelledanti-digoxigenin antibody solution) with the antibody dilution (preparedwith quantum sufficient of sterilized-purified water to be 0.7 ml astheir total volume containing 8.48 mg ofTris-(Hydroxymethyl)-Aminomethane, 6.14 mg of Sodium Chloride, andquantum sufficient of Hydrochloric Acid), then 10 μl of the labelledantibody solution is dropped onto each of the smeared area, and they maybe left for from 15 to 60 minutes. After then, they are immersed in fromtwo-fold to fifty-fold diluted solution, preferably ten-fold dilutedsolution of the labelled antibody detergent solution (prepared withsterilized-purified water to be 100 ml as their total volume containing1 ml of Polysolvate 20 and 50 ml of PBS Raw Solution) and arecentrifuged for from five to 30 minutes during which they are beingimmersed in the detergent solution. This operation is repeated twice,then the samples are immersed in the treatment solution prepared bymixing Preliminary Treatment Solution 1 (prepared with quantumsufficient of sterilized-purified water to be 50 ml as their totalvolume containing 6.06 g of Tris-(Hydroxymethyl)-Aminomethane, 2.92 g ofSodium Chloride, and quantum sufficient of Hydrochloric Acid) with equalamount of Preliminary Treatment Solution 2 (prepared with quantumsufficient of sterilized-purified water to be 50 ml as their totalvolume containing 5.08 g of Magnesium Chloride Hexahydrate), thendiluting approximately 5-fold with sterilized-purified water, and arecentrifuged for from five to 30 minutes during which they are beingimmersed in the detergent solution. 1 ml of chromogenic agent (NitroBlue Tetrazolium (NBT)/5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) persingle slide is dropped onto the smeared area of the slides byfiltrating with a disposable syringe equipped with 0.2 μm syringe topfilter, and is kept in the dark and is left in a wet chamber for fromabout 15 minutes to about 60 minutes at from about 10° C. to about 45°C., preferably at 37° C. Then, they are immersed for from about two toabout 10 minutes in the solution prepared by diluting from two-fold toabout 50-fold, preferably about ten-fold the chromogenic agent cleaner(prepared with quantum sufficient of sterilized-purified water to be 50ml as their total volume containing 606 mg ofTris-(Hydroxymethyl)-Aminomethane, quantum sufficient of HydrochloricAcid and 186 mg of Disodium Ethylenediaminetetraacetic acid Dihydrate)and are air-dried. Further, they are immersed in the solution preparedby diluting from two-fold to 50-fold, preferably 10-fold thecounterstain solution (prepared with quantum sufficient ofsterilized-purified water to be 50 ml as their total volume containing50 mg of Fast Green FCF (Edible Dye Green No. 3)) and in an acetic acidsolution of from about 0.1% to about 5%, preferably about 1%.Thereafter, they may be immersed again in the solution prepared bydiluting about two-fold to about 50-fold, preferably 10-fold the cleaneraforenoted to remove excessive counterstain solution and are completelyair-dried. Each of such chromogenic agents may be prepared individually.

Preferable solution of the anti-digoxigenin antibodies labelled withalkaline phosphatase may include a solution which will offer color inthe blotted area made by blotting one ng of DNA labelled withdigoxigenin onto a blotting membrane, blocking the same, then treatingthose with the 10,000-fold diluted solution of the anti-digoxigeninantibody labelled with alkaline phosphatase, and reacting those withchromogenic substrates (NBT/BCIP), while it will not offer any coloraccording to the same procedure employing DNA without digoxigenin label.Anti-digoxigenin antibodies taken from sheep are preferable. Inparticular, it is recommended to take such antibodies by purifying theimmunized sheep serum with an ion-exchange chromatography and anantibody column chromatography.

Chromogenic agent (NBT/BCIP solution, pH 9.0-10.0) is an agent preparedpreferably with quantum sufficient of sterilized-purified water to be 10ml as their total volume containing 3.3 mg of Nitro Blue Tetrazolium(NBT), 1.65 mg of 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP), 99 μg ofN,N-dimethyl-formamide, 121 mg of Tris-(Hydroxymethyl)-Aminomethane,quantum sufficient of Hydrochloric Acid, 58.4 mg of Sodium Chloride,101.6 mg of Magnesium Chloride Hexahydrate.

Preferable chromogenic agents may include an agent which will offerviolet signals in the blotted area made by blotting proteins labelledwith alkaline phosphatase onto a blotting membrane and treating the samewith such chromogenic agents in the dark at room temperature.

In the counterstain above, edible dye, for example, Yellow No. 4(Tartrazine) can be used to present more clearly contrast betweensignals and cells. Factors of such poor counter contrast may includesimilarity of the colors to be expressed, namely, between violet coloroffered by the substrate and blue color offered by Naphthol Black. Whenthis methodology is applied to the present invention, it came to knowthat such methodology is useful at the counterstain. Any conventionalmethodology has never employed a food dye.

Nick translation methodology is applicable as a method to label thedigoxigenin. The other methodology may include, for example, PCR Method,Random Primer Labelling Method, in vitro Transcription Labelling Method,Terminal Transferase Labelling Method or the like.

When at least one expressed violet signal(s) is/are confirmed by theoptical microscopy (×1,000) on the subjected cells in the single wellwhich is stained with the counterstain solution aforenoted, the samplemay be designated as positive.

Then, Japanese Patent Nos. 2558420, 2798499, 2965543, 2965544 and3026789 can be referred at the producing of the detective probes.

For example, in order to culture bacteria taken from working-cell-banks,the working-cell-banks (SA-24) are smeared streakily with a platinumloop, a disposable plastic loop or the like onto L-broth solid mediumcontaining 50 μg/ml ampicillin mounted on the sterilized laboratorydishes (Screening).

After an overnight cultivation thereof, single colony so cultured isinoculated into 5 ml of L-broth medium containing 50 μg/ml ampicillinand is shaking cultured overnight at 37° C. (Precultivation).

2.5 ml of the culture solution is inoculated individually into 400 ml ofthe medium in a flask and is shaking cultured overnight at about 37° C.(Main Cultivation).

Then, in order to extract SA-24 plasmid DNA, the cultured solutionprepared through the main cultivation are centrifugated at 4° C., in4,000×g, for 10 minutes to collect the bacteria. Supernatant areremoved, 20 ml of STE (10 mmol/l Tris-HCl (pH 8.0), 1 mmol/l DisodiumEthylenediaminetetraacetic acid (EDTA), 0.1 mmol/l Sodium Chloride) areadded to the remained bacteria to resuspend the same, and they arecentrifugated at 4° C., in 4,000×g, for 10 minutes to collect thebacteria. The bacteria is suspended with 5 ml of Solution-1 (50 mmol/lglucose, 25 mmol/l Tris-HCl (pH 8.0), 10 mmol/l EDTA) containing 10mg/ml Lysozyme and is left for five minutes under the room temperature.Thereafter, 10 ml of Solution-2 (0.2 mmol/l Sodium Hydroxide, 1% SodiumDodecyl Sulfate (SDS)) is added, then they are upset to mix, and areleft for ten minutes on ice. 7.5 ml of Iced Solution-3 (3 mol/lpotassium acetate (pH 4.8)) is added, then they are upset to mix, andare left for ten minutes on ice.

After centrifugation by a high speed refrigerated centrifuge at 4° C.for 30 minutes at 45,000×g, the supernatant is recovered, and left tostand to cool to room temperature. After leaving to stand, 0.6 volume ofisopropanol (about 24 ml) is added thereto, mixed by inversion and leftto stand at room temperature for 5 minutes or longer. Aftercentrifugation by a high speed refrigerated centrifuge at 25° C., for 30minutes at 28,000×g, the supernatant is discarded, and thus resultingpellet is washed with 70% ethanol and air dried. After air drying, 8 mlof TE (10 mmol/l Tris-hydrochloric acid (pH 8.0), 1 mmol/l EDTA) isadded thereto to dissolve the pellet (extraction of plasmid DNA).

Next, for the purification of the plasmid DNA containing SA-24, 800 μlof 10 mg/ml ethidium bromide and 8.6 g of cesium chloride are added tothe resulting plasmid DNA followed by mixing by inversion to dissolvethe plasmid. The solution is placed in a centrifuge tube, which is thencapped or sealed. After centrifugation at 20° C. for 5 hours at500,000×g with a vertical rotor, a band of the plasmid DNA isfractionated using a glass syringe or an injection needle under theirradiation of an ultraviolet ray light. To the fractionated plasmid DNAsolution is added an equivalent amount of TE-saturated 1-butanolfollowed by mixing by inversion and centrifugation at 15,000×g for 5minutes by a high speed microcentrifuge to remove the supernatant. Thisoperation is repeated to eliminate ethidium bromide in the plasmid DNAsolution. Next, thereto is added TE to give the volume of 1.5 mlfollowed by desalting on a demineralization column (NAP-10). To thedesalted plasmid DNA solution is added 30 μl of a 3 mol/l sodium acetatesolution followed by mixing, and 3 fold amount of 99.5% ethanol is addedthereto followed by mixing by inversion and leaving to stand at −20° C.for 30 minutes or longer. After leaving to stand, centrifugation isconducted with a high speed refrigerated micro centrifuge at 4° C. for20 minutes at 15,000×g to remove the supernatant. Thereafter, cold 70%ethanol is added thereto to suspend therein, and once again,centrifugation is conducted with a high speed refrigerated microcentrifuge at 4° C. for 20 minutes at 15,000×g to remove thesupernatant. Thus resulting precipitate of the plasmid DNA is evaporatedto dryness under a reduced pressure. TE in an amount of 100 μl is addedto the plasmid DNA to dissolve completely, and the concentration ismeasured on the basis of the absorbance at 260 nm (Purification ofplasmid DNA containing SA-24). Then, size check of the plasmid DNAcontaining SA-24 is carried out by a treatment with a restriction enzymeand agarose electrophoresis.

In order to purify SA-24 by digesting the plasmid DNA containing SA-24with restriction enzyme(s) and successively applying them to an agaroseelectrophoresis, 1 mg of the plasmid DNA with SA-24, molecular weight ofwhich had been determined, are digested through a reaction to beperformed at 37° C. for one and half hours or more under the presence ofthe restriction enzyme HindIII alone or the combination of HindIII andthe other restriction enzyme(s). After digesting the plasmid DNA,completion of such digestion is confirmed by applying a part of thereactionary solution to 0.8% agarose electrophoresis. Then, afterconfirming such digestion, SA-24 band is taken by applying the solutionto an electrophoresis with 0.8% agarose as a fractionator. SA-24 sotaken are extracted from the agarose gel and are purified, then,concentration thereof is determined with an absorptiometer. A part ofthe purified SA-24 is applied to an electrophoresis with 0.8% agarosegel and is appeared as a single band.

SA-24 may preferably be labelled with digoxigenin with the reactionarysolution having the composition listed in the following Table 1including 2 μg of the purified SA-24.

TABLE 1 Composition in Reactionary Solution to Label Amount(μL) DNAprobe X 10 × L.B.^((a)) 10 100 mmol/L dithiothreitol 10 dNTPs^((b)) (A,G, C 0.5 mmol/L) 4 digoxigenin-dUTP^((c)) (0.4 mmol/L) 5 DNAseI^((d)) 610 U/μL DNA polymerase I 2 Sterilized-Purified Water Y Total 100[REMARKS] ^((a))10 × L.B.: 0.5 mol/L Tris-HCl (pH 7.5), 50 mmol/Lmagnesium chloride, 0.5 mg/mL bovine serum albumin ^((b))dNTPs: 0.5mmol/L 2′-dioxyadenosine-5′-triphosphate, 0.5 mmol/L2′-dioxyguanosine-5′-triphosphate, 0.5 mmol/L2′-dioxycytidine-5′-triphosphate ^((c))digoxigenin-dUTP: 0.4 mmol/Ldigoxigenin-11-2′-dioxy-uridine-5′-triphosphate ^((d))DNAseI:Deoxyribonuclease I is diluted with 25 mmol/L Tris-HCl (pH 7.5), 50%glycerin solution to be used at 50–150 mU per total amount of 100 μL andis adjusted to the amount aforenoted.

In Table 1, the volume X is adjustable to realize preferable probeconcentration aforenoted according to the concentration of raw probesolution, then, the final volume is adjusted by determining, based onthe volume X, the volume Y of Sterilized-Purified Water. After puttingthe labels, the reaction is terminated by adding 100 μl of TE to thereaction solution.

Free nucleotides are then removed by adding solution to terminate thereaction into the spin column and centrifuging the same at 4° C., in380×g, for 10 minutes. Thereafter, concentration of the eluted solutionis determined with an absorptiometer and is adjusted with TE to the unitlevel of ng/μl.

To confirm the labelled subjects, 0.5 μl of the labelled SA-24 isdropped onto a membrane and is air-dried. The membrane is immersed intothe blocking reagent and is kept for 30 minutes under the roomtemperature. The membrane is then immersed for 30 minutes under the roomtemperature into the solution of anti-digoxigenin antibody labelled withalkaline phosphatase prepared by diluting 5,000-fold with a solutioncontaining 0.1 mol/l Tris-HCl (pH 7.5) and 0.15 mol/l sodium chloride.The membrane is immersed into a solution containing 0.1 mol/l Tris-HCl(pH 7.5) and 0.15 mol/l sodium chloride, then is shaked for 10 minutesunder the room temperature, and is rinsed twice. Further, the membraneis immersed for 10 minutes under the room temperature into a solutioncontaining 0.5 mol/l Tris-HCl (pH 9.5), 0.15 mol/l sodium chloride and50 mmol/l magnesium chloride. The membrane is then immersed intochromogenic agent for 10 minutes, in the dark, under the roomtemperature. Labelled subjects are confirmed with violet color appearedunder the spotted position.

Spin column is produced by filling a few amount of sterilized grass woolinto 1 ml volume of disposable syringe. Sephadex G-50 swelled with 1mmol/l Tris-HCl (pH 7.5), 1 mmol/l EDTA and 0.1% SDS is then filled intothe syringe. The syringe is inserted into 15 ml volume of the disposableconical tube and is centrifuged at 4° C., in 320×g, for 10 minutes toremove the excess buffer solution. The syringe is then detached from thedisposable conical tube and the excess buffer solution is discarded.Thereafter, the spin column is assembled by engaging 1.5 ml volume ofEppendolf Tube with the bottom of the disposable conical tube andentering the syringe into the conical tube.

Dot Blot Hybridization according to the following procedure isrecommended to confirm specificity of the probes.

First of all, in order to denature each of the spotted genomic DNA,according to the standard procedure, 100 ng of the various bacterialgenome so prepared are spotted onto nylon membranes (Pall BioDine TypeB; Nihon Pall Ltd.), and air-dried membranes are kept for 10 minutes onthe filter papers (3 mm: Wattman) saturated with solution containing 0.5mol/l sodium hydroxide and 1.5 mol/l sodium chloride. The denatured DNAare then neutralized by leaving them for 10 minutes on the previouslynoted filter papers saturated with solution containing 0.5 mol/lTris-HCl (pH 7.5) and 1.5 mol/l sodium chloride. They are further leftfor five minutes on the previously noted filter papers saturated with2×SSC (Standard Saline Citrate) Solution and are rinsed. After then,such membranes are air-dried and are immersed into 2×SSC Solution forfive minutes. In accordance with the standard procedure, the membranesare immersed into Prehybridization Solution in plastic bags and are keptat 42° C. for 60 minutes. In the plastic bag, the membranes are immersedin 15 ml of Hybridization Solution containing 400 ng of Probes and arereacted overnight at 42° C. Next, the membranes are immersed into thesolution containing 2×SSC and 0.1% SDS (sodium dodecyl sulfate) and arerinced for five minutes (this is repeated twice). The membranes are thenimmersed into the solution containing 0.1×SSC, 0.1% SDS and are rincedfor ten minutes (this is repeated three times). The membranes areimmersed into 2×SSC solution and are rinced for five minutes. Themembranes are immersed into the solution containing 3% bovine serumalbumin, 1% Blocking Buffer (Boehringer), 0.1 mol/l Tris-Hcl (pH 7.5)and 0.15 mol/l Sodium Chloride, and are gently shaked for 30 minutes.The membranes are immersed into the solution prepared by diluting5,000-fold the alkaline-phosphatase labelled anti-digoxigenin antibody(Boehringer) with the solution containing 0.1 mol/l Tris-HCl (pH 7.5)and 0.15 mol/l Sodium Chloride, and are gently shaked for 30 minutes.Next, the membranes are immersed into the solution containing 0.1 mol/lTris-HCl (pH 7.5) and 0.15 mol/l sodium chloride, and are shaked for 15minutes (twice). The membranes are immersed into the solution containing0.1 mol/l Tris-HCl (pH 9.5), 0.1 mol/l Sodium Chloride and 5 mmol/lMagnesium Chloride, and are shaked for 5 minutes. The membranes areimmersed into NBT-BCIP Solution (GIBCO BRL) and are subjected tochromogenic reaction in the dark. The membranes are then immersed intoTE (10 mmol/l Tris-HCl (pH 8.0), 1 mmol/l EDTA) to terminate thechromogenic reaction and are air-dried. Particulars of PrehybridizationSolution and Hybridization Solution are illustrated in the followingTable 2.

TABLE 2 Prehybridization Hybridization Solution Solution Formamide 7.56.75 20 × SSC Solution 3.75 3.75 100 × Denhart Solution 0.75 0.15 0.5mol/L Phosphatized 0.75 0.6 Buffer Solution Sterized-Distillated Water1.5 1.95 10 mg/mL Salmon Sperm DNA 0.75 0.3 50% Dextran Sulfate — 1.5Total Liquid Amount 15.0 15.0 [Unit: ml]

Conventional surfactants can be employed as those to be used in the stepof in situ hybridization. Typically, the surfactants are roughly dividedinto anionic surfactant, non-ionic surfactant, cationic surfactant andamphoteric surfactant.

Anion surfactants are also referred to as anionic surfactants, whichyield an organic anion upon ionization in water. When a lipophilic groupin the molecule of the surfactant is represented by R, examples of theanion surfactant include RCOONa, RSO₃Na, RSO₄Na and the like. An aqueoussolution of the surfactant containing a weakly acidic group such asRCOONa is liable to be hydrolyzed and is weak alkaline. However, anaqueous solution of a surfactant having a strongly acidic group such asRSO₃Na, RSO₄Na or the like is resistant to hydrolysis, which shall beneutral. Because it is anionic, it may lose surface activity in thepresence of a large quantity of cationic substance, and may beinactivated in a strongly acidic circumstance.

Nonionic surfactants refer to those having a hydrophilic group which isnonionic. An ethylene oxide group (—CH₂CH₂O—) is often used as thehydrophilic group. As number of this group increases, hydrophilicity isincreased. To the contrary, as number of the lipophilic group increases,lipophilicity is increased. Therefore, it is characterized in thatsurfactants with variously altered hydrophilicity and lipophilicity canbe obtained. Because a nonionic surfactant does not ionize in water andis hardly affected by inorganic salts, less action is exerted also on aliving body. In addition, the detergent action thereof is potent withcomparatively less foaming, therefore, it is widely used not alone as adetergent, but in pharmaceuticals, cosmetics, foods and the like. Watersoluble nonionic surfactant becomes insoluble in water at a certaintemperature as the temperature rises, and then the aqueous solutionstarts to be turbid. Such turbidity results from the cleavage ofhydrogen bonds between the hydrophilic groups and water.

Cation surfactants are also referred to as cationic surfactants, whichyield an organic cation upon ionization in water. Although cationsurfactants do not have potent detergent action in general, theystrongly bind to anionic substances such as bacteria, leading to a greatbactericidal action. Moreover, they also have an anti-static ability forfibers and plastics. Although dodecyltrimethyl chloride[C₁₂H₂₅(CH₃)₃N]Cl as a typical exemplary cation surfactant is watersoluble, didodecyldimethylammonium chloride [(C₁₂H₂₅)₂(CH₃)₂N]Cl isinsoluble in water, which forms a vesicle in the form of a bimolecularfilm in water, and is soluble in benzene.

Ampholytic surfactants are surfactants having both an anionic group anda cationic group in the molecule. Ionization state thereof in water issimilar to those of amino acids, and thus many of ampholytic surfactantsare amino acid derivatives. Therefore, they have an isoelectric pointsimilarly to amino acids, which act as an anion surfactant in analkaline region from the isoelectric point, whilst as a cationsurfactant in an acidic region. Water solubility becomes the lowest atthe isoelectric point, and the surface tension is also reduced.Ampholytic surfactants are used for a bactericide, an antistatic agentor the like.

Furthermore, anion surfactants are classified into the carboxylic acidtype, sulfonic acid type, sulfate ester type and phosphate ester type,whilst nonionic surfactants are classified into the ester type, ethertype, ester ether type and alkanolamide type. Cation surfactants areclassified into alkylamine salt type and quaternary ammonium salt type,whilst ampholytic surfactants are classified into carboxy betaine type,2-alkylimidazoline derivative type and glycine type.

Moreover, the anion surfactants of carboxylic acid type are furtherclassified into fatty acid monocarboxylate salts, N-acylsarcosine saltsand N-acylglutamate salts. Representative examples thereof respectivelyinclude: sodium laurate and medicated soap as the fatty acidmonocarboxylate salts; sodium N-lauroylsarcosine as the N-acylsarcosinesalt; and disodium N-lauroylglutamate as the N-acylglutamate. Stillmore, the sulfonic acid type is further classified into dialkylsulfosuccinate salts, alkane sulfonate salts, alpha-olefin sulfonatesalts, straight chain alkyl benzenesulfonate salts, alkyl (branchedchain) benzenesulfonate salts, alkyl naphthalenesulfonate salts,naphthalenesulfonate salts-formaldehyde condensates andN-methyl-N-acyltaurine salts. Representative examples include: sodiumdioctyl sulfosuccinate as the dialkyl sulfosuccinate salt; sodiumdodecane sulfonate as the alkane sulfonate; sodium straight chaindodecyl benzenesulfonate as the straight chain alkyl benzenesulfonatesalt; sodium dodecyl benzenesulfonate as the alkyl (branched chain)benzenesulfonate salt; sodium butyl naphthalenesulfonate as the alkylnaphthalenesulfonate salt; and sodium N-methyl-N-stearoyltaurine as theN-methyl N-acyltaurine salt. In addition, the sulfate ester type isfurther classified into alkyl sulfate salts, polyoxyethylene alkyl ethersulfate salts and oil-and-fat sulfate ester salts. Representativeexamples include sodium dodecyl sulfate, sodium lauryl sulfate andsodium cetyl sulfate as the alkyl sulfate salt; and polyoxyethylenelauryl ether sulfate triethanolamine as the polyoxyethylene alkyl ethersulfate salt. Moreover, the phosphate ester type is further classifiedinto alkyl phosphate salts, polyoxyethylene alkyl ether phosphate saltsand polyoxyethylene alkylphenyl ether phosphate salts. Representativeexamples include disodium monolauryl phosphate as the alkyl phosphatesalt; and sodium polyoxyethylene lauryl ether phosphate andpolyoxyethylene oleyl ether phosphate (8 MOL) as the polyoxyethylenealkyl ether phosphate salt.

Ester type of the nonionic surfactants is further classified into fattyacid glycerin, fatty acid sorbitan and fatty acid sucrose ester.Representative examples respectively include: glycerin monostearate asthe fatty acid glycerin; sorbitan monostearate, sorbitan trioleate,sorbitan sesquioleate, sorbitan monolaurate, polysorbate 20(polyoxyethylene sorbitan fatty acid ester), polysorbate 60 andpolysorbate 80 as the fatty acid sorbitan; and stearic acid sucroseester as the fatty acid sucrose ester. Additionally, the ether type isfurther classified into polyoxyethylene alkyl ether, polyoxyethylenealkyl phenyl ether and polyoxyethylene polyoxypropylene glycol.Representative examples include: polyoxyethylene lauryl ether,polyoxyethylene stearyl ether and polyoxyethylene cetyl ether as thepolyoxyethylene alkyl ether; and polyoxyethylene nonyl phenyl ether andpolyoxyethylene octyl phenyl ether as the polyoxyethylene alkyl phenylether. In addition, the ester ether type is further classified intofatty acid polyethylene glycol and fatty acid polyoxyethylene sorbitan.Representative examples thereof respectively include oleic acidpolyethylene glycol as the fatty acid polyethylene glycol; andpolyoxyethylene sorbitan palmitate and polyoxyethylene sorbitanmonolaurate as the fatty acid polyoxyethylene sorbitan. In addition, thealkanolamide type involves only fatty acid alkanolamide alone.Representative example is lauric diethanolamide.

The alkyl amine salt type of the cation surfactant includes monoalkylamine salts, dialkyl amine salt and trialkyl amine salts. Representativeexamples thereof include monostearyl amine hydrochloride. Moreover, thequaternary ammonium salt type is further classified into alkyltrimethylammonium chloride (or bromide or iodide), dialkyldimethyl ammoniumchloride (or bromide or iodide), and alkyl benzalkonium chloride.Representative examples respectively include: stearyltrimethyl ammoniumchloride as the alkyltrimethyl ammonium chloride (or bromide or iodide);distearyldimethyl ammonium chloride as the dialkyldimethyl ammoniumchloride (or bromide or iodide); and lauryl benzalkonium chloride as thealkyl benzalkonium chloride.

The carboxy betaine type of the ampholytic surfactant is only alkylbetaine alone. Representative example is lauryl betaine. Additionally,the 2-alkyl imidazoline derivative type is only2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine alone.Representative example includes 2-undecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine. In addition, the glycine type may be alkyl (ordialkyl) diethylene triaminoacetic acid, and the representative exampleincludes dioctyl diethylene triaminoacetic acid.

Moreover, in addition to the representative examples as described above,Triton X-100, lauryl sarcosine, saponin, BRIJ35, alkyl allyl polyetheralcohol, higher alcohol sulfate, N-cocoyl-L-arginine ethyl esterDL-pyrrolidone carboxylate salt, sodium N-cocoyl-N-methyl aminoethylsulfonate, cholesterol, self emulsifying type monostearate glycerin,squalane, stearyl alcohol, stearic acid polyoxyl 40, cetyl alcohol,cetomacrogol 1000, sebacate diethyl, nonylphenoxy polyoxyethylene ethanesulfate ester ammonium, polyoxyethylene oleylamine, polyoxyethylenesorbit yellow bees wax, polyoxyl 35 castor oil, macrogol 400, N-coconutoil fatty acid acyl L-arginine ethyl-DL-pyrrolidone carboxylate salt,lauryldimethylamine oxide solution, lauromacrogol, methylcellulose, CMC(carboxymethylcellulose), polyoxyethylene hardened castor oil 20 andpolyoxyethylene hardened castor oil 60, CHAPS, deoxycholic acid,digitonin, n-dodecyl maltoside, Nonidet P40, n-octyl glucoside, octylthioglucoside, laurate sucrose, dodecyl poly(ethylene glycolether)n,n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and the likeare also included.

It is important to apply the various surfactants listed above at the insitu hybridization step, while application embodiment thereof is notlimited anyway. For example, one skilled in the art would mix thesurfactants previously into probe solution or probe dilution, otherwise,the skilled artisan may prepare the separate solution containingsurfactants and add the same before, simultaneously or after applicationof the probe solution onto the area to be smeared.

If any positive control probe is necessary in the present invention,such probe may be prepared as follows. For example, in order to conductthe extraction and purification of the genomic DNA of U937 cell (ATCCCRL-1593.2), U937 cells are first cultured in a 5% carbon dioxide gasincubator at 37° C. using an RPMI1640 medium (25 ml) in a cell cultureflask (175 cm²). The U937 culture solution is placed in a 50 mlcentrifuge tube, and centrifuged at 4° C. for 10 minutes at 220×g torecover the U937 cells. The cells are suspended and washed in 10 ml ofPBS, and again centrifuged at 4° C. for 10 minutes at 180×g to recoverthe cells. Thereafter, the supernatant is discarded, and the cells aresuspended in 1 ml of a TE solution containing 200 μg/ml proteinase K andcontaining 1% SDS, followed by leaving to stand at 37° C. for 30minutes. Phenol extraction is repeated three to four times to executedeproteinization. Genome deposited through the ethanol precipitation isrecovered, dissolved in 500 μl of sterile purified water containing 2.5μg of ribonuclease, and left to stand at 42° C. for 30 minutes. Thephenol extraction is repeated two to three times to executedeproteinization. Genome deposited through the ethanol precipitation isrecovered, and dissolved in 500 μl of TE. Thereafter, a positive controlprobe can be produced by measuring the concentration with an absorbancemeter, and subjecting to digoxigenin labelling. Moreover, the positivecontrol probe which may preferably used is one which permits toascertain the hybrid formation when the positive control probe issubjected to dot hybridization on a membrane with 100 ng of U937 genomespotted thereon. When a negative control probe is required, it can beproduced by any known method.

Further, the present invention also includes a kit for detecting and/oridentifying causative microorganisms of infectious diseases by takingphagocytes from the clinical specimens containing active phagocytes,immobilizing the phagocytes so taken, treating the phagocytes to improvecell membrane permeabilities thereof, further treating the phagocytes tobare DNA in the causative microorganisms which might be existed in thephagocytes, in situ hybridizing DNA so bared with detective DNA probe(s)which can hybridize with such bared DNA under stringent conditions, anddetecting and/or identifying the causative microorganisms based onsignals so detected, the kit comprises at least one or more enzymeselected from the group consisting of lysostafin, lysozyme,N-acetylmuramidase and zymolase used in the exposing step of the DNA,probe solution containing surfactant, and one or more DNA probe fordetection. The kit includes, reagent for separating blood, enzymepretreatment reagent, enzyme reagent, acetylation reagent, probesolution, blocking reagent, labelled antibody, labelled antibodydiluent, coloring pretreatment liquid-1, coloring pretreatment liquid-2,coloring reagent, counter staining solution, PBS stock solution,hybridization stock solution, labelled antibody washing solution,coloring reagent washing solution, APS coated slide glass, probedilution solution, buffer A and the like as demonstrated in thefollowing Examples. Among these, it is preferred that at least theenzyme reagent and the probe solution are included. In addition, variousreagents used in the present invention may be included for example,chloroform, ethanol, acetic anhydride, DMSO, PMSF, formamide, aceticacid, hydrochloric acid, sodium hydroxide and the like. Moreover,instrument and machine such as low speed centrifuge, incubator, countingchamber, shaker, humid box, incubator, light microscope, variablepipette, blood collection tube, tip, pipette, staining bottle, measuringcylinder, glass syringe, 0.2 μm syringe top filter may be included.

Furthermore, the present invention provides a process for monitoring thegene of a foreign microorganism digested by a phagocyte included in aclinical specimen which contains a phagocyte derived from a living body.Moreover, the present invention provides a process for identifying thegene of a microorganism which becomes a candidate of the causativemicroorganism which a causative microorganism of sepsis or a causativemicroorganism of bacteriemia is specified on the basis of the resultsidentified.

It was revealed that when this process was applied in practice todiagnoses for blood of a variety of patients suspected as suffering fromsepsis, causative microorganism could be detected with about four timeshigher sensitivity compared to the blood culture process with noinfluence of the administered antimicrobial agent, and the identity ofthe detected microorganism strain was favorable. Furthermore, incomparison with the blood culture which requires three days or longerand approximately 14 days for the examination, an accurate result can beachieved by a simple operation within a short time period, i.e., about 8hours, until the completion of the entire operation, according to theprocess of the present invention, an useful marker can therefore berealized in monitoring and the like in prognosis or diagnosis of aninfectious disease such as sepsis, bacteriemia or the like in which arapid and favorable care is particularly necessary.

EXAMPLES

The present invention will be described in detail along with thefollowing Examples, but as a matter of course would not be limited tothe Examples because of the following disclosures.

Example 1 Blooding and Preparation of Blood-Specimens

Twelve blood specimens (Specimen A-L) were obtained as a clinicalspecimen from the patients who are under the suspicion about sepsis. 10ml of heparinized venous bloods were obtained from each patient and weremixed with the blood components separative reagent (adjusted withsterilized-purified water to be 25 ml as their final volume containing225 mg of Sodium Chloride and 1.5 g of Dextran (Molecular Weight:200,000-300,000)) in the ratio of 4:1. Leukocyte fractions (the upperlayer) were then obtained by leaving them at 37° C. for 30 minutes.Leukocytes were appeared by centrifuging the leukocyte fractions in160×g, at 4° C. and for 10 minutes. Pelletized leukocytes so obtainedwere suspended with 1 ml of sterilized-purified water and the suspensionwere immediately put into an isotonic state by adding thereto excessiveamounts of PBS (prepared by diluting twenty-fold withsterilized-purified water the PBS Raw Solution which have been adjustedwith sterilized-purified water to be 120 ml as their final volumecontaining 18.24 g of Sodium Chloride, 6.012 g of Sodium MonohydrogenPhosphate Didecahydrate and 1.123 g of Sodium Dihydrogen PhosphateDihydrate). Such suspension were then re centrifuged in 160×g, at 4° C.and for 10 minutes.

Example 2 Fixation of Leukocytes

APS coated slides were employed wherein they were made by coating3-aminopropyltriethoxysilane (APS, SIGMA) onto the slides (JAPAN ARBROWN CO., LTD., PRODUCTS ID. S311BL). Namely, such APS coated slideswere produced by putting the slides (PRODUCTS ID. S311BL) into holders,immersing them into a diluted neutral detergent to wash the same,removing the detergent well therefrom with tap water then with purifiedwater, leaving them under the higher temperature (100° C. or more) todry the slides, and leaving the same under the room temperature to coolthe slides. After then, these slides were immersed in Acetone containing2% APS for one minute and were immediately rinsed gently with Acetonethen with sterilized-purified water. These slides were then air-dried.Such performance was repeated and includes the immersion of these slidesin Acetone containing 2% APS for one minute, immediate gentle rinse ofthe slides with Acetone and sterilized-purified water, and air-dry ofthe same. Finally, the slides were dried under the temperature of 42° C.to realize the APS coated slides.

Pelletized leukocytes were prepared by centrifuging leukocyte fractionsin 160×g, at 4° C. and for 10 minutes. Then, leukocyte population wasdetermined by adding small amount of PBS to the pelletized leukocytes,suspending the same, and counting the population with a hemacytometer.Leukocytes were duly mounted on the APS coated slides by smearing 5 μlof the leukocyte suspension into each well of the slides adjusted withPBS to be cell population of 1×10⁵ cells/well, realizing the extendedmono-layer of the leukocytes, and completely air-drying the same. Afterthen, they were immersed in Carnoy s fixative solution (prepared bymixing Ethanol, Chloroform and Acetic Acid in the volume ratio of 6:3:1)for 20 minutes and were immersed in 75% Ethanol solution for fiveminutes. Finally, they were completely air-dried.

Example 3 Treatment to Improve Permeability of Leukocyte Cell Membrane

They were immersed in PBS for 10 minutes, then in the solution preparedby diluting ten-fold a pretreatment reagent (prepared by mixing 1.25 gof Saponin, 1.25 ml t-octylphenoxy-polyethoxyethanol (specific gravityof 1.068-1.075 (20/4° C.), pH (5 w/v %) 5.5-7.5) and 25 ml PBS RawSolution, and adjusting with sterilized-purified water to be 50 ml astheir final volume), and were applied to a centrifuge for 10 minutes toaccelerate their permeability.

Example 4 Treatment of Bacterial Cell Wall with Lytic Enzymes

In order to bare DNA in the causative microorganisms, an enzyme solutionwas prepared by adding, per single slide, 1 ml of a reagent solvent(prepared by diluting 100-fold, with PBS, Dimethyl Sulfoxide (DMSO)containing 0.1 mol/l Phenylmethyl Sulfonylfluoride (PMSF)) to an enzymereagent (N-acetylmuramidase 1,000 Units/ml, Lysozyme 100,000 Units/mland/or Lysostaphin 100 Units/ml) then 1 ml of which were dropped underthe temperature of 37° C.-42° C. in the wet chamber onto the area wherethe leukocytes were smeared and were left for 30 minutes. Then, theywere immersed in PBS containing 0.2 mol/l Hydrochloric Acid (prepared byadding hydrochloric acid to PBS Raw Solution, diluting 20-fold the samewith sterilized-purified water and adjusting final concentration ofHydrochloric Acid to 0.2 mol/l), and were applied to a centrifuge for 10minutes to accelerate their permeability.

Example 5 Acetylation of Cell Membrane Proteins

Slides were then immersed in the acetylation reagent prepared by addingAcetic Anhydrate to the raw acetylation reagent (adjusted with quantumsufficient of sterilized-purified water to be 50 ml as their totalvolume containing 7.46 g of Triethanolamine and quantum sufficient ofHydrochloric Acid), diluting 10-fold with sterilized-purified water, andadjusting final concentration of Acetic Anhydride to 0.8%, and wereapplied to a centrifuge for 10 minutes. After then, they were completelyair-dried by immersing them successively into 75%, 85% and 98% Ethanolfor three minutes respectively.

Example 6 Alkalization of Bacterial DNA Denaturation of Double-StrandedDNA into Single-Stranded DNA

Slides were then immersed for three minutes in PBS containing 70 mmol/lSodium Hydroxide (prepared by adding Sodium Hydroxide to PBS RawSolution, diluting 20-fold with sterilized-purified water, and adjustingfinal concentration of Sodium Hydroxide to 70 mmol/l). After then, theywere completely air-dried by immersing them successively into 75%, 85%and 98% Ethanol for three minutes respectively.

Example 7 Hybridization

Probe Solution (1.0 ng/μl) containing 15 ng of DNA probes labelled withdigoxigenin prepared with the diluted probe solution (containing 0.25%SDS, 600 μl of Salmon Sperm DNA, 50 μl of 100×Denhurt's Solution, 500 μlof Hybridization Raw Solution, 2250 μl of Formamide and 1000 μl of 50%Dextran Sulfate) was applied to the smeared area and was left for twohours under the temperature of 37° C.-42° C. in the wet chamber. ProbeSolution without SDS was employed as a control. DNA probes labelled withdigoxigenin were prepared through nick translation methodology. Then,three stained bottles containing the hybridization detergent (preparedby mixing Hybridization Raw Solution (prepared with sterilized-purifiedwater to be 75 ml as their total volume containing 13.15 g of SodiumChloride, 6.615 g of Trisodium Citrate Dihydrate) in the ratio ofHybridization Raw Solution:sterilized-purified water:Formamide=5:45:50)were provided and they were successively immersed therein at 42° C. for10 minutes respectively.

After then, they were centrifuged for 10 minutes during which they werebeing immersed in PBS. As DNA probes labelled with digoxigenin, probesfrom Staphylococcus aureus of SA-24 (SEQ ID NO: 1), SA-36 (SEQ ID NO: 2)and SA-77 (SEQ ID NO: 3) as well as those from Staphylococcusepidermidis of SE-22 (SEQ ID NO: 4), SE-3 (SEQ ID NO: 5) and SE-32 (SEQID NO: 6) (See, Japanese Patent No. 2798499) were employed. Also, as aprobe from Pseudomonas aeruginosa, that of P2-2 (SEQ ID NO: 7) (See,Japanese Patent No. 2965544) was employed. Then, as probes fromEnterococcus faecalis, those of EF-1 (SEQ ID NO: 8), EF-27 (SEQ ID NO:9) and EF-7 (SEQ ID NO: 10) (See, Japanese Patent No. 2965543) wereemployed. Then, probes from Escherichia coli of EC-24 (SEQ ID NO: 11),EC-34 (SEQ ID NO: 12) and EC-39 (SEQ ID NO: 13), that from Enterobactercloacae of ET-49 (SEQ ID NO: 14), and that from Klebsiella pneumoniae ofKI-50 (SEQ ID NO: 15) (See, Japanese Patent No. 3026789) were alsoemployed. Further, as probes from Candida albicans, those of CA-26 (SEQID NO: 16), CA-26-1 (SEQ ID NO: 17), CA-26-2 (SEQ ID NO: 18) and CA-26-3(SEQ ID NO: 19) (See, Japanese Patent No. 2558420) were employed. Thepresent probes were produced with the probes listed above throughnick-translation methodologies.

Example 8 Blocking

After in situ hybridization, blocking operation was performed. 1 ml ofBlocking reagent (prepared with sterilized-purified water to be 10 ml astheir final volume containing 2 ml of Rabbit Normal Serum and 0.5 ml ofPBS Raw Solution) per single slide was dropped onto the smeared areathereof and the slides were left for 30 minutes. Then, Blocking reagentwas removed.

Example 9 Reaction with Labelled Antibody

Labelled antibody solution was prepared by diluting 50-fold the labelledantibody (prepared with 12.6 μl of Buffer A (prepared with quantumsufficient of sterilized-purified water to be 100 ml as their totalvolume containing 746 mg of Triethanolamine, 17.5 mg of Sodium Chloride,20.3 mg of Magnesium Chloride Hexahydrate, 1.36 mg of Zinc Chloride,1000 mg of Bovine Serum Albumin and quantum sufficient of HydrochloricAcid) to be 14 μl as their total volume containing 1.05 Unit ofalkaline-phosphatase-labelled anti-digoxigenin antibody solution) withthe antibody dilution (prepared with quantum sufficient ofsterilized-purified water to be 0.7 ml as their total volume containing8.48 mg of Tris-(Hydroxymethyl)-Aminomethane, 6.14 mg of SodiumChloride, and quantum sufficient of Hydrochloric Acid), then 10 μl ofthe labelled antibody solution were dropped onto each of the smearedarea, and they were left for 30 minutes. After then, they were immersedin the ten-fold diluted solution of the labelled antibody detergentsolution (prepared with sterilized-purified water to be 100 ml as theirtotal volume containing 1 ml of Polysolvate 20 and 50 ml of PBS RawSolution) and were centrifuged for 10 minutes during which they werebeing immersed in the detergent solution. This operation was repeatedtwice, then the samples were immersed in the treatment solution preparedby mixing Preliminary Treatment Solution 1 (prepared with quantumsufficient of sterilized-purified water to be 50 ml as their totalvolume containing 6.06 g of Tris-(Hydroxymethyl)-Aminomethane, 2.92 g ofSodium Chloride, and quantum sufficient of Hydrochloric Acid) with equalamount of Preliminary Treatment Solution 2 (prepared with quantumsufficient of sterilized-purified water to be 50 ml as their totalvolume containing 5.08 g of Magnesium Chloride Hexahydrate) and diluting5-fold with sterilized-purified water. The samples were then centrifugedfor 10 minutes during which they were being immersed in the treatmentsolution.

Example 10 Detection

1 ml of chromogenic agent (Nitro Blue Tetrazolium(NBT)/5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) Solution, pH 9.0-10.0:prepared with quantum sufficient of sterilized-purified water to be 10ml as their total volume containing 3.3 mg of NBT, 1.65 mg of BCIP, 99μg of N,N-dimethyl-formamide, 121 mg ofTris-(Hydroxymethyl)-Aminomethane, quantum sufficient of HydrochloricAcid, 58.4 mg of Sodium Chloride, 101.6 mg of Magnesium ChlorideHexahydrate) per single slide was dropped onto the smeared area of theslides by filtrating with a disposable syringe equipped with 0.2 μmsyringe top filter, and was kept in the dark and was left for 30 minutesat 37° C. in a wet chamber. Then, they were immersed for five minutes inthe solution prepared by diluting ten-fold the chromogenic agent cleaner(prepared with quantum sufficient of sterilized-purified water to be 50ml as their total volume containing 606 mg ofTris-(Hydroxymethyl)-Aminomethane, quantum sufficient of HydrochloricAcid and 186 mg of Disodium Ethylenediaminetetraacetic acid Dihydrate)and were air-dried. Further, they were immersed in the solution preparedby diluting 10-fold the counterstain solution (prepared with quantumsufficient of sterilized-purified water to be 50 ml as their totalvolume containing 50 mg of Fast Green FCF (Food Dye Green No. 3)) and inthe 1% acetic acid solution. Thereafter, they were immersed again in thesolution prepared by diluting 10-fold the cleaner aforenoted to removeexcessive counterstain solution and were completely air-dried.

Example 11 Criterion

When at least one expressed violet signal(s) is/are confirmed by theoptical microscopy (×1,000) on the subjected cells which were stainedwith the counterstain solution in single well, the sample was designatedas positive. As the result of the present method, of twelve specimens,bacteria had been detected in the five specimens of Specimen A-SA(Staphylococcus aureus), Specimen F and G-SE (Staphylococcusepidermidis), Specimen J-SE and EF (Enterococcus faecalis), and SpecimenL-SA and CA (Candida albicans). In the meantime, when the same specimenswere subjected to the hemoculture methodologies according to the knownprocedures, although the identical result was confirmed in Specimen A asa detection of SA, no bacteria had been detected in any of Specimen F,G, J and L. Accordingly, it became apparent that the present methodcould detect those rapidly with better sensitivity in comparison withthe hemoculture methodologies.

With regard to the result on Specimen A-SA, the effect offered by addingSDS to the probe dilution is shown in FIG. 1. Obviously from FIG. 1,detection sensivity on the signal could be improved remarkably withaddition of 0.25% SDS. Turning to the other specimens, signals couldsimilarly be detected clearly by adding SDS thereto. Meanwhile, theprobes employed in this Example were those produced by performing nicktranslation on a combination of the base sequences from SA-24 (SEQ IDNO:1), SA-36 (SEQ ID NO:2) and SA-77 (SEQ ID NO:3).

Example 12 Investigation on Optimum Cell Population of Leukocytes to beSmeared and Mounted

Optimum Cell Population of Leukocytes to be smeared onto the well (thecircular well of 5 mm diameter) of APS coated slides had beeninvestigated. 10 ml of heparinized healthy human bloods were collected,then, the leukocytes were obtained therefrom according to the proceduresof Example 1. Leukocytes so obtained were suspended in the quantumsufficient of PBS and the leukocyte population per 1 ml was determinedwith a hematimeter. A serial dilution commencing at (a) 1×10⁸ cells/mland followed by (b) 5×10⁷ cells/ml, (c) 1×10⁷ cells/ml, (d) 5×10⁶cells/ml, (e) 1×10⁶ cells/ml, (f) 5×10⁵ cells/ml and (g) 1×10⁵ cells/mlwas prepared and 5 μl of each population were smeared onto the slides.

Smeared slides were air-dried and were fixed with Carnoy s fixativesolution (See, Example 2). Immediately thereafter, they were stainedwith the counterstain solution aforenoted and were subjected to thecriterion according to the method of Example 11. As a result thereof,the cell population of 1×10⁸ cells/ml was too many to detect and was notappropriate. Then, the cell populations of 5×10⁶ cells/ml or less werenot appropriate, because little population were observed in the well.Accordingly, phagocyte density (X cells/ml) to be immobilized should beadjusted to the range of about 5×10⁶ cells/ml<X cells/ml<about 1×10⁸cells/ml, preferably, to that of about 1×10⁷ cells/ml≦X cells/ml≦about5×10⁷ cells/ml. It was concurrently apparent therefrom that leukocytepopulation (y cells/well (5 mm diameter)) to be immobilized in thesingle well of APS coated slides should be adjusted to the range ofabout 2.5×10⁴ cells/well<y cells/well (5 mm diameter)<about 5×10⁵cells/well, preferably, about 5×10⁴ cells/well≦y cells/well (5 mmdiameter)≦about 2.5×10⁵ cells/well. Experimental results on the samples(a)-(f) were illustrated in FIG. 2 (a)-(f) respectively.

Example 13 Investigation of Lytic Enzyme to be Employed

Enzymatic conditions to lyse Staphylococcus aureus (ATCC 12600),Staphylococcus epidermidis (ATCC 14990), Pseudomonas aeruginosa (ATCC10145), Enterococcus faecalis (ATCC 19433) and Escherichia coli (ATCC11775) had been investigated. Lysostaphin (Bur. J. Biochem., 38, pp.293-300, 1973) was used as an lytic enzyme for Staphylococcus aureus andStaphylococcus epidermidis. As for Enterococcus faecalis,N-acetylmuramidase (Archs. Oral Biol., 23, pp. 543-549, 1978), Lysozyme(SEIKAGAKU CORPORATION) was used. Then, as for Pseudomonas aeruginosaand Escherichia coli, PBS containing 70 mmol/l sodium hydroxide wasused. All of these bacteria were inoculated into 5 ml of BHI (BrainHeart Infusion) liquid medium (DIFCO) and were cultivated under 37° C.for eight hours or more. Cultivated solution were collected bycentrifuging them in 2,000×g, at 4° C., for 10 minutes. Samples to besubjected were prepared by suspending the collected bacteria in PBS.

Lysis activities were determined with a microplate reader by evaluatingdecreased density at 600 nm absorbance on the subjected sample solution.As a result thereof, Staphylococcus aureus and Staphylococcusepidermidis were lysed with Lysostaphin. As for Pseudomonas aeruginosaand Escherichia coli, enzyme treatment was not necessary, because theywere lysed with PBS containing 70 mmol/l sodium hydroxide. Then, as forEnterococcus faecalis, it was discovered that lysis activities thereforwere improved by not relying on N-acetylmuramidase alone but on thecombination of N-acetylmuramidase with Lysozyme. But, for example, whenPseudomonas aeruginosa, Escherichia coli or the like are the digestedbacteria with phagocytosis, such enzyme treatments may not be necessary,because their cell walls are solved at the alkaline treatment, thereby,their genes are bared. Each enzyme to be employed in the pretreatmentprocedure of the present invention for solving the exogenousmicroorganism would be valid not only for the previously noted bacteriastrain, but also for the other bacterial strains including thosebelonged to Staphylococcus genus, Streptococcus genus, Bacillus genusand Micrococcus genus. Such enzymes can be used as an individual enzyme,but to employ them as the mixed enzymes is quite useful. These resultswere illustrated in FIG. 3, in particular, as FIG. 3 (a) Staphylococcusaureus and Staphylococcus epidermidis, (b) Pseudomonas aeruginosa andEscherichia coli, and (c) Enterococcus faecalis.

Example 14 Investigation of Enzyme Solution Investigation of OptimumConcentration of DMSO

Since protease contained in the enzyme reagent degrades a form ofleukocyte, enzyme activity of DMSO which is solvent of PMSF to be usedto keep the form of leukocyte had been investigated. Enterococcusfaecalis was inoculated in 50 ml of BHI liquid medium noted previouslyand was cultivated, at 37° C., for eight hours or more. Bacterialcells/were collected by centrifuging the cultivation liquid, at 4° C.,in 2,000×g, for 10 minutes, then were suspended in PBS and weresubjected to heat-treatment in an autoclave (120° C., 10 minutes). Next,these were centrifuged, at 4° C., in 2,000×g, for 10 minutes, thendiscarding the supernatant, and the precipitates were suspended in 1 mlof PBS and were lyophilized. The lyophilized samples were suspended in 5mmol/l Tris-HCl (pH 6.0) containing 0-10% DMSO then 2 mmol/l MagnesiumChloride and were designated as a sample on N-acetylmuramidase.Micrococcus luteus (JCM1464) was inoculated in 5 ml of BHI liquid medium(supra) and was cultivated, at 37%, for eight hours or more. Bacterialcells/were collected by centrifuging the cultivated bacterial solution,at 4° C., in 2,000×g, for 10 minutes. Then the supernatant wasdiscarded, then, bacterial cells/were collected by rinsing the bacterialpellets though suspension of them in PBS and recentrifuging the same, at4° C., in 2,000×g, for 10 minutes. The bacteria so collected weresuspended in PBS containing 0-10% DMSO and were designated as a sampleon Lysozyme. On the other hand, samples on Lysostaphin were alsoprepared by culturing and collecting Staphylococcus epidermidissubstantially along with the procedure for the samples on Lysozyme, andsuspending the cultured bacteria with PBS containing 0%˜10% DMSO.

Enzymatic activities were evaluated by determining, with a micro platereader, decrease of absorbance at 600 nm on the subjected samples.Correlation between DMSO and the enzymatic activities were experimentedin this example under the enzymatic titer of (a) N-acetylmuramidase 300Units/ml, (b) Lysozyme 10,000 Units/ml and (c) Lysostaphin 50 Units/ml.When each enzymatic activity was evaluated with decrease of the densityof bacteria suspension (O.D.=600 nm) per predetermined time, there islittle correlation between DMSO and N-acetylmuramidase activity, while5% or more of DMSO lowered activities of Lysozyme and Lysostaphin. Therewas no decrease on the enzymatic activities in DMSO concentration of 2%or less. Accordingly, concentration of DMSO to solve PMSF is adjusted toless that 5%, preferably 2% or less, more preferably about 1%. Resultswere shown in FIG. 4 (a)-(c) and in the following Table 3.

TABLE 3 Correlation between DMSO and Enzymatic Activity (DensityDecrease in Bacteria Suspension) Amounts of DMSO AddedN-acetylmuramidase Lysozyme Lysostaphin (%) O.D./5 minutes O.D./3minutes O.D./10 minutes 0 (control) 79.3 ± 4.8 0.689 ± 0.028 0.168 ±0.017 0.1 75.0 ± 3.2 0.678 ± 0.026 0.164 ± 0.009 1 75.8 ± 2.8 0.660 ±0.026 0.160 ± 0.008 2 75.8 ± 2.5 0.653 ± 0.024 0.145 ± 0.009 5 76.3 ±4.9 0.566 ± 0.017 0.124 ± 0.006 10 73.8 ± 3.5 0.464 ± 0.016 0.094 ±0.006

Example 15 Examination on Enzymatic Lysis Solution Examination onOptimal Concentration of PMSF

Because protease included in the enzyme reagent deteriorates themorphology of leukocytes, effects of PMSF (manufactured by PIERCE),which is added for the purpose of retaining the morphology of theleukocytes, on enzymatic activity were examined. PMSF was dissolved in100 μl of DMSO (manufactured by Wako Pure Chemical Industries, Ltd), anddiluted to 10 ml with PBS such that the final concentration of PMSFbecomes none (0 mmol/l) to 1 mmol/l. To this solution was addedproteinase K (manufactured by Boeringer Mannheim) such that titer of theprotease becomes 0.2 unit/ml. Heparinized healthy human blood in anamount of 5 ml was collected, and leukocytes were obtained according tothe process described in Example 1. Next, the leukocytes were suspendedin an appropriate amount of PBS, and the cell number was measured usinga counting chamber. Cell number was adjusted to about 5×10⁴ cells/wellto about 2.5×10⁵ cells/well, and 5 μl therefrom was smeared on the wellof the APS coated slide glass. After air drying, fixation was executedaccording to the method of Carnoy fixation described in Example 2. Usingthis sample, tests were performed according to the process described inExamples 3 to 11. As a consequence of performing the tests at theconcentration of PMSF of 1 μmol/l to 1 mmol/l, effects were found at theconcentration of 10 μmol/l or greater, while deterioration of morphologyof the leukocytes was completely suppressed at the concentration of PMSFof 0.1 mmol/l or greater. The results are shown in FIG. 5, for (a):protease 0.2 unit/ml alone, (b): 1 μmol/ml PMSF added, (c): 10 μmol/mlPMSF added, (d): 0.1 mmol/ml PMSF added, and (e): 1 mmol/ml PMSF added,respectively.

Example 16 Examination of Optimal Titer of Lytic Enzyme, Zymolase

Optimal titer of zymolase for exposing DNA was examined through lysis ofCandida albicans. Candida albicans was inoculated in YPD medium, andcultured over day and night at 30° C. Then, two types of the solutionswere prepared: a solution of Candida albicans as a substrate suspendedin PBS (substrate 1); and a solution prepared by Carnoy s fixation,immersing in 70% ethanol, air drying and suspension in PBS (substrate2). Upon the reaction, a mixture of zymolase/PBS: 0.5 ml, substrate: 1.5ml, M/15 phosphate buffer: 2.5 ml and sterile purified water: 0.5 ml,adjusted to give the total volume of 5.0 ml was used.

Thereafter, the reaction was allowed at 37° C. for 2 hours, and theOD₈₀₀ was measured. Furthermore, the concentration of zymolase(Zymolyase-100T) for use was 0 mg/ml, 0.01 mg/ml, 0.025 mg/ml, 0.05mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 2.5 mg/ml and 5 mg/ml.Consequently, each OD₈₀₀ value when the substrate 1 was used was 0.533,0.521, 0.553, 0.554, 0.548, 0.417, 0.394, 0.288, 0.163 and 0.113, andeach OD₈₀₀ value when the substrate 2 was used was 0.445, 0.411, 0.359,0.282, 0.232, 0.146, 0.115, 0.096, 0.08 and 0.057. It was proven thateffectiveness was brought when both of the substrate 1 and substrate 2were in the range of 0.5 mg/ml to 5 mg/ml, and particularly 1 mg/ml to 5mg/ml. That is, the amount of zymolase to be used is preferably 50unit/ml to 500 unit/ml, particularly 100 unit/ml to 500 unit/ml.

Example 17 Examination of Optimal Condition (Titer) of EnzymaticTreatment

(1) Production of Digested Sample

[1] Preparation of U937 Cell

U937 cells (monocyte established cell line: ATCC CRL-1593.2) werecultured in an RPMI 1640 medium (25 ml) within a cell culture flask (175cm²) in a 5% carbon dioxide gas incubator at 37° C. Next, the U937 cellculture liquid was placed in a 50 ml centrifuge tube, and centrifuged at4° C. for 10 minutes at 220×g to recover the U937 cells. Then, thusrecovered U937 cells were suspended in 200 μl of PBS, and the cellnumber was counted with a counting chamber. The cell number was adjustedto 1×10⁴ cells/μl to 2×10⁴ cells/μl.

[2] Preparation of Bacterial Digested Sample

Staphylococcus aureus (ATCC 12600), Staphylococcus epidermidis (ATCC14990), Pseudomonas aeruginosa(ATCC 10145), Enterococcus faecalis (ATCC19433) and Escherichia coli (ATCC 11775) were inoculated in each 5 ml ofBHI culture medium, and cultured at 37° C. for 8 hours or longer. Thecultured bacterial liquid was centrifuged at 4° C. for 10 minutes at2,000×g to collect the bacteria. After discarding the supernatant, thebacterial pellet was suspended in 5 ml of PBS, and centrifugation wasconducted once again at 4° C. for 10 minutes at 2,000×g to collect thebacteria. Thus collected bacteria were suspended in 5 ml of PBS andthereafter, 15 ml of bacterial liquids was produced prepared by dilutingin PBS to give the turbidity (O.D.=600 nm) of the bacterial liquid,which was measured with a absorbance meter, of 0.01 to 0.03 forStaphylococcus aureus, 0.01 to 0.03 for Staphylococcus epidermidis, 0.02to 0.03 for Pseudomonas aeruginosa, 0.01 to 0.03 for Enterococcusfaecalis, 0.02 to 0.03 for Escherichia coli, respectively. Thus producedbacterial liquid was transferred into a separate 175 cm² flask forculture, and left to stand still at room temperature for 30 minutes.Fifty ml of heparinized healthy human blood was collected, and theretowas added the reagent for separating haemocyte at a ratio of 4:1, andleft to stand still at 37° C. for 30 minutes to yield the leukocytefraction. Thus obtained leukocyte fraction was adjusted to 50 ml withPBS. The supernatant in the culture flask (supra) was gently discarded,and each 10 ml of the leukocyte fraction diluted in PBS was added to theflask followed by leaving to stand still at room temperature for 10minutes. The supernatant in the flask for culture was discarded, and theleukocytes attached to the bottom of the flask were recovered in a 15 mlcentrifuge tube with 10 ml of PBS containing 0.02% EDTA, and centrifugedat 4° C. for 10 minutes at 140×g to 180×g to collect the leukocytes.Because contamination of erythrocytes was found in the collectedleukocytes, precipitates of the leukocytes were gently suspended in 1 mlof sterile purified water to allow hemolysis, subjected to isotonizationthrough adding 14 ml of PBS, followed by centrifugation once again at 4°C. for 10 minutes at 140×g to 180×g to collect the leukocytes. Thecollected leukocytes were suspended in PBS, and cell number was countedwith a counting chamber to adjust to give 1×10⁴ cells/μl to 5×10⁴cells/μl. These digested samples were referred to as SA digested sample,SE digested sample, PA digested sample, EF digested sample and EKdigested sample.

[3] Smear and Fixation

Each 5 μl of U937 cells prepared in Example 17 (1) [1] and each 5 μl ofeach bacterial digested sample produced in Example 17 (1) [2] weresmeared on each well of the APS coated slide glass, and air dried. Next,after immersing the slide glass in the Carnoy s fixative described inExample 2 for 20 minutes, it was immersed in 75% ethanol for 5 minutes.After washing Carnoy's fixative and air drying, the slide glass wasstored at 4° C. until use in the test (see, Example 2). Next,pretreatment of the fixed sample was carried out according to Example 3.

(2) Standard and Process for Testing Digested Sample

[1] Cell Number

Cell number to be smeared and fixed on the slide glass of each bacterialdigested sample was 5.0×10⁴ to 2.5×10⁵ cells/well, whilst cell number ofU937 cells was 5.0×10⁴ to 1.0×10⁵ cells/well.

[2] Phagocytosis Rate

The bacterial digested sample smeared and fixed on the slide glass wasstained with an acridine orange staining solution, and about 200 cellswere randomly counted with a fluorescence microscope (×1,000). Among themeasured cells, cells including bacteria phagocytized within the cells(cells with any change characteristic in phagocytosis found inmorphology, as shown by arrows in FIG. 6) were determined as positivecells, and the phagocytosis rate (%) was calculated according to themathematical formula below.Phagocytosis rate (%)=[(Positive cell number/Measured cell number)×100]

Thus calculated phagocytosis rate (%) of each bacterial digested samplewas 10% or greater.

[3] Test Process

The digested sample produced in Example 17 (2) [1] and [2] was employedas a specimen. The SA digested sample used had the phagocytosis rate of23% with 1.98×10⁵ cells/well. The SE digested sample had thephagocytosis rate of 27% with 1.74×10⁵ cells/well. Moreover, the EFdigested sample had the phagocytosis rate of 34% with 6.40×10⁴cells/well.

Using the slide glass having each digested sample smeared thereon, theenzymatic pretreatment was performed according to the process describedin Example 3. Next, the slide glass after completing the enzymaticpretreatment was placed in a humid box, and the reaction was allowed bydropping 1 ml of each enzyme solution prepared to give each titer on thesmeared site of the specimen. Thereafter, the slide glass was immersedin PBS containing 0.2 mol/l hydrochloric acid, and in 70% ethanolrespectively, for 10 minutes, and air dried. After immersing this slideglass in PBS containing 70 mmol/l sodium hydroxide for 3 minutes, and in70% ethanol for 10 minutes, it was air dried and stained with 1%acridine orange staining solution. Then, evaluation was made with afluorescence microscope (X 1,000). For Staphylococcus aureus andStaphylococcus epidermidis, examination of the optimal titer wasconducted with lysostafin. In order to examine the optimal titer whenN-acetylmuramidase and lysozyme are used in combination for Enterococcusfaecalis, examination on optimal titer of lysozyme was conducted incases where N-acetylmuramidase was fixed at 100 unit/ml, and on optimaltiter of N-acetylmuramidase in cases where lysozyme was fixed at 10,000unit/ml. The determination was made as adequate when no bacterial bodywas identified in the leukocytes by the enzymatic treatment.

[4] Results

For the lysis of Staphylococcus aureus, as described in Table 4,sufficient effects were exerted at the titer of lysostafin of 1 unit/ml,however, upon lysis of Staphylococcus epidermidis, the titer oflysostafin of 10 unit/ml or greater was necessary. Therefore, theoptimal titer of lysostafin was set to be 10 unit/ml to 100 unit/ml. Inaddition, for the lysis of Enterococcus faecalis, lysis did not occurwith the titer of N-acetylmuramidase of 10 unit/ml or less when thetiter of lysozyme was fixed at 10,000 unit/ml. In respect of lysozyme,when the titer of N-acetylmuramidase was fixed at 100 unit/ml, lysis didnot occur with the titer of lysozyme of 1,000 unit/ml or less, asdescribed in Table 5. Therefore, the optimal titer of N-acetylmuramidasewas set to be 100 unit/ml to 1,000 unit/ml, whilst the optimal titer oflysozyme was set to be 10,000 unit/ml to 100,000 unit/ml. The resultsare shown in FIG. 7. In the Figure, depicted are states of: (a) thedigested sample of Staphylococcus aureus prior to the enzymatictreatment, (b) the digested sample of Enterococcus faecalis prior to theenzymatic treatment, (c) the sample (a) following the enzymatictreatment, and (d) the sample (b) following the enzymatic treatment.

TABLE 4 Optimal Titer for Enzymatic Treatment of Lysostafin on S.Aureus, S. epidermidis (U/mL) Digested Lysostafin Titer Samples 0 0.1 110 100 1,000 SA once inadequate Inadequate adequate adequate adequateadequate Digested twice inadequate inadequate adequate adequate adequateadequate Sample thrice inadequate inadequate adequate adequate adequateadequate SE once inadequate inadequate inadequate adequate adequateadequate Digested twice inadequate inadequate inadequate adequateadequate adequate Sample thrice inadequate inadequate inadequateadequate adequate adequate

TABLE 5 Optimal Titer of Enzymatic Treatment of N-acetylmuramidase andlysozyme on E. faecalis titer (U/mL) Digested N-acetyl muramidase Sample0 1 10 100 1,000 10,000 EF once Inadequate inadequate inadequateadequate adequate adequate Digested twice Inadequate inadequateinadequate adequate adequate adequate Sample thrice Inadequateinadequate inadequate adequate adequate adequate (U/mL) DigestedLysozyme titer Sample 0 10 100 1,000 10,000 100,000 EF once Inadequateinadequate inadequate inadequate adequate adequate Digested twiceInadequate inadequate inadequate inadequate adequate adequate Samplethrice Inadequate inadequate inadequate inadequate adequate adequate

Applications of these results obtained using the digested samples to thepresent invention could result in similar results. Therefore, theoptimal titer of each enzyme as described above in the identification ofa causative microorganism of an infectious disease in the clinicalspecimen of the present invention was also set similarly.

Example 18 Examination on Optimal Condition of Enzymatic Treatment(Temperature)

Using a slide glass including each digested sample smeared thereon,examination was conducted according to the process described in example17 (2) [3]. Time period of the enzymatic treatment in this test was 30minutes, and the temperature for examination was 4° C., 25° C., 37° C.,42° C., and 60° C. Moreover, titer of each enzyme was N-acetylmuramidase(100 unit/ml, manufactured by Seikagaku Corporation), lysozyme (10,000unit/ml, manufactured by Seikagaku Corporation), lysostafin (10 unit/ml,manufactured by SIGMA).

Determination was conducted according to the process described inexample 17 (2) [3]. As a consequence, for Staphylococcus aureus, nobacterial body was found in the leukocytes in the range of temperatureof 4° C. to 60° C. For Staphylococcus epidermidis, although bacterialbodies remained in the leukocytes at the temperature of 4° C. and 25°C., no bacterial body was found at 37° C. or higher. Further, forEnterococcus faecalis, although bacterial bodies remained at thetemperature of treatment of 4° C., 25° C. and 60° C., no bacterial bodywas found at 37° C. and 42° C. Hence, the optimal temperature for theenzymatic treatment was set to be 37° C. to 42° C. The results are shownin Table 6.

TABLE 6 Optimal Temperature for Treatment of Enzyme Reagent DigestedTemperature for Treatment (° C.) Samples 4 25 37 42 60 SA Once adequateadequate adequate adequate adequate Digested twice adequate adequateadequate adequate adequate Sample thrice adequate adequate adequateadequate adequate SE once inadequate inadequate adequate adequateadequate Digested twice inadequate inadequate adequate adequate adequateSample thrice inadequate inadequate adequate adequate adequate EF onceinadequate inadequate adequate adequate inadequate Digested twiceinadequate inadequate adequate adequate inadequate Sample thriceinadequate inadequate adequate adequate inadequate

Applications of these results obtained using the digested samples to thepresent invention could result in similar results. Therefore, theoptimal temperature of the enzymatic treatment in the identification ofa causative microorganism of an infectious disease in the clinicalspecimen of the present invention was also set similarly.

Example 19 Examination on Optimal Condition of Enzymatic Treatment(Time)

Digested samples produced according to the process described in Example17 (1) [1] and [2] were used as specimens. Time period of theexamination was 0 minute, 10 minutes, 20 minutes, 30 minutes, 60 minutesand 120 minutes. Phagocytosis rate of the used SA digested sample was18% with 7.80×10⁴ cells/well. Phagocytosis rate of the used SE digestedsample was 34% with 1.10×10⁵ cells/well. Further, phagocytosis rate ofthe EF digested sample was 28% with 1.30×10⁵ cells/well.

Using the slide glass including each digested sample smeared thereon,examination was conducted according to the process described in example17 (2) [3]. Temperature for the enzymatic treatment in this test was 37°C., and titer of each enzyme was 100 unit/ml for N-acetylmuramidase,10,000 unit/ml for lysozyme, 10 unit/ml for lysostafin. Determinationwas conducted according to the process described in example 17 (2) [3].As a consequence, for all of Staphylococcus aureus, Staphylococcusepidermidis and Enterococcus faecalis digested samples, no bacterialbody was found in the leukocytes with the time period of the enzymatictreatment of 20 minutes or longer (inadequate at 0 minute and 10minutes). Therefore, the optimal time period of the enzymatic treatmentis at least 15 minutes or longer, preferably 20 minutes or longer, andstill preferably 30 minutes to 60 minutes. The results are shown inTable 7.

TABLE 7 Optimal Time Period of Treatment of Enzyme Reagent Digested Timeof enzyme-treatment (minutes) Samples 0 10 20 30 60 120 SA onceinadequate inadequate adequate adequate adequate adequate Digested twiceinadequate inadequate adequate adequate adequate adequate Sample thriceinadequate inadequate adequate adequate adequate adequate SE onceinadequate inadequate adequate adequate adequate adequate Digested twiceinadequate inadequate adequate adequate adequate adequate Sample thriceinadequate inadequate adequate adequate adequate adequate EF onceinadequate inadequate adequate adequate adequate adequate Digested twiceinadequate inadequate adequate adequate adequate adequate Sample thriceinadequate inadequate adequate adequate adequate adequate

Applications of these results obtained using the digested samples to thepresent invention could result in similar results. Therefore, theoptimal time period of the enzymatic treatment in the identification ofa causative microorganism of an infectious disease in the clinicalspecimen of the present invention was also set similarly.

Example 20 Examination on Optimal Condition of Enzymatic Treatment(Time)

In in situ hybridization reaction according to the present invention,concentration of the probe is an important factor which affects thehybridizing velocity. When the probe concentration is too low, thereaction velocity may be lowered, leading to the possibility of unclearsignal. Furthermore, use of an excess amount of probe may result ingrounds for nonspecific reaction.

Thus, optimal concentration was examined in connection with variousprobe solutions. First, the digested samples produced according to theprocess described in Example 17(1) [1] and [2] were used as specimens.The phagocytosis rate of the used SA digested sample was 24% with1.48×10⁵ cells/well. The phagocytosis rate of the SE digested sample was28% with 2.07×10⁵ cells/well. The phagocytosis rate of the PA digestedsample was 11% with 1.59×10⁵ cells/well. In addition, the phagocytosisrate of the EF digested sample was 24% with 1.72×10⁵ cells/well. Thephagocytosis rate of the EK digested sample was 12% with 1.63×10⁵cells/well. Using the slide glass including each digested sample smearedthereon, examination was conducted according to the process described inExample 17(2) [3]. The probes for use were labelled with digoxigenin,and the concentration of each probe for Staphylococcus aureus,Staphylococcus epidermidis, Enterococcus faecalis, Pseudomonasaeruginosa and Escherichia coli was adjusted to 0.06 ng/μl, 0.6 ng/μl,1.2 ng/μl, 1.8 ng/μl, 2.4 ng/μl, 3 ng/μl, respectively. The probesolution prepared to each concentration described above was used on theslide glass including the digested sample smeared thereon (see, FIG. 8),and examined according to the process described in Examples 3-11.

Consequently, the signal became unclear at lower concentration (0.06ng/μl), and on the other hand, increase in background was observed athigher concentration (2.4 ng/μl and 3 ng/μl). Therefore, theconcentrations of probes of SA, SE, PA, EF and EK were determined to be0.6 to 1.8 ng/μl, preferably 0.6 to 1.2 ng/μl. Moreover, since aninadequate result was yielded at 0.06 ng/μl, while an adequate resultwas yielded at 0.6 ng/μl, it is preferably determined to be 0.1 ng/μl orgreater.

Furthermore, since an inadequate result was yielded at 2.4 ng/μl, and anadequate result was yielded at 1.8 ng/μl, it is preferably determined tobe 2.2 ng/μl or less. The results are shown in Tables 8-12 below.

TABLE 8 SA probe Probe concentration (ng/μL) Digested sample 0.06 0.61.2 1.8 2.4 3 SA digested sample − + + + + + SE digested sample − − −− + + PA digested sample − − − − + + EF digested sample − − − − + + EKdigested sample − − − − + +

TABLE 9 SE probe Probe concentration (ng/μL) Digested sample 0.06 0.61.2 1.8 2.4 3 SA digested sample − − − − − + SE digested sample− + + + + + PA digested sample − − − − − + EF digested sample − − − −− + EK digested sample − − − − − +

TABLE 10 PA probe Probe concentration (ng/μL) Digested sample 0.06 0.61.2 1.8 2.4 3 SA digested sample − − − − − − SE digested sample − − −− + + PA digested sample − + + + + + EF digested sample − − − − − + EKdigested sample − − − − − +

TABLE 11 EF probe Probe concentration (ng/μL) Digested sample 0.06 0.61.2 1.8 2.4 3 SA digested sample − − − − − + SE digested sample − − −− + + PA digested sample − − − − + + EF digested sample − + + + + + EKdigested sample − − − − − −

TABLE 12 EK probe Probe concentration (ng/μL) Digested sample 0.06 0.61.2 1.8 2.4 3 SA digested sample − − − − + + SE digested sample − − −− + + PA digested sample − − − − + + EF digested sample − − − − + + EKdigested sample − + + + + +

Applications of these results obtained using the digested samples to thepresent invention could result in similar results. Therefore, theoptimal concentration of each probe described above in theidentification of a causative microorganism of an infectious disease inthe clinical specimen of the present invention was also set similarly.

Example 21 Examination on Hybridization Temperature

Reaction temperature in the hybridization reaction is a parameter whichaffects the hybridizing velocity and stability of the hybrid. Becausehigh temperature of the hybridization reaction is known to deterioratethe cell morphology, examination of the optimal temperature (4° C., 25°C., 37° C., 42° C., 50° C. and 60° C.) was performed.

First, the digested samples produced according to the process describedin Example 17(1) [1] and [2] were used as specimens. The phagocytosisrate of the used SA digested sample was 31% with 1.38×10⁵ cells/well.The phagocytosis rate of the SE digested sample was 42% with 1.95×10⁵cells/well. The phagocytosis rate of the PA digested sample was 14% with1.27×10⁵ cells/well. In addition, the phagocytosis rate of the EFdigested sample was 48% with 1.05×10⁵ cells/well. The phagocytosis rateof the EK digested sample was 17% with 1.85×10⁵ cells/well.

Using the slide glass including the digested sample and U937 cellssmeared and fixed thereon (see, FIG. 9), examination was conductedaccording to the process described in Examples 3-11. Consequently, nostable signal was observed for each type of probe at the hybridizationtemperature of 4° C. or less owing to the lowered hybridizationvelocity. Further, at 60° C., changes in cell morphology were detected,and thus no stable signal was observed. In addition, at 25° C. and 50°C., detection could be executed better compared to at the temperature of37° C. and 42° C., although the signal was unclear. Hence, optimaltemperature of the hybridization may be 25° C. to 50° C., morepreferably 37 to 42° C. The results are shown in Tables 13-17 below.

TABLE 13 SA probe Hybridization temperature (° C.) Digested sample 4 2537 42 50 60 SA digested sample − + + + + + SE digested sample − − − − −− PA digested sample − − − − − − EF digested sample − − − − − − EKdigested sample − − − − − −

TABLE 14 SE probe Hybridization temperature (° C.) Digested sample 4 2537 42 50 60 SA digested sample − − − − − − SE digested sample + + + + +− PA digested sample − − − − − − EF digested sample − − − − − − EKdigested sample − − − − − −

TABLE 15 PA probe Hybridization temperature (° C.) Digested sample 4 2537 42 50 60 SA digested sample − − − − − − SE digested sample − − − − −− PA digested sample − + + + + − EF digested sample − − − − − − EKdigested sample − − − − − −

TABLE 16 EF probe Hybridization temperature (° C.) Digested sample 4 2537 42 50 60 SA digested sample − − − − − − SE digested sample − − − − −− PA digested sample − − − − − − EF digested sample + + + + + − EKdigested sample − − − − − −

TABLE 17 EK probe Hybridization temperature (° C.) Digested sample 4 2537 42 50 60 SA digested sample − − − − − − SE digested sample − − − − −− PA digested sample − − − − − − EF digested sample − − − − − − EKdigested sample − + + + + −

Applications of these results obtained using the digested samples to thepresent invention could result in similar results. Therefore, theoptimal temperature of hybridization in the identification of acausative microorganism of an infectious disease in the clinicalspecimen of the present invention was also set similarly.

Example 22 Examination on Hybridization Time Period

The digested samples produced according to the process described inExample 17(1) [1] and [2] were used as specimens, and examination wasconducted on the time period of hybridization of 10 minutes, 60 minutes,90 minutes, 120 minutes, 180 minutes and 900 minutes. The phagocytosisrate of the used SA digested sample was 47% with 1.45×10⁵ cells/well.The phagocytosis rate of the SE digested sample was 47% with 1.33×10⁵cells/well. The phagocytosis rate of the PA digested sample was 15% with1.91×10⁵ cells/well. In addition, the phagocytosis rate of the EFdigested sample was 41% with 1.45×10⁵ cells/well. The phagocytosis rateof the EK digested sample was 20% with 1.23×10⁵ cells/well.

Using the slide glass including the digested sample and U937 cellssmeared and fixed thereon (same as one shown in FIG. 9), examination wasconducted according to the process described in Examples 3-11.Consequently, although no signal was observed with the time period ofhybridization of 10 minutes, a signal was observed at 60 minutes orgreater, and a stable signal was observed at 90 minutes or greater.Further, no alteration in detection of the signal was observed also withthe time period of hybridization of 900 minutes. Therefore, it ispreferred that the time period is at least 30 minutes or greater,preferably 60 minutes or greater, and more preferably 90 minutes orgreater. More preferred optimal time period of hybridization may be setto be 120 minutes to 900 minutes. The results are shown in Tables 18-22below.

TABLE 18 SA probe Hybridization time (minutes) Digested sample 10 60 90120 180 900 SA digested sample − + + + + + SE digested sample − − − − −− PA digested sample − − − − − − EF digested sample − − − − − − EKdigested sample − − − − − −

TABLE 19 SE probe Hybridization time (minutes) Digested sample 10 60 90120 180 900 SA digested sample − − − − − − SE digestedsample + + + + + + PA digested sample − − − − − − EF digested sample − −− − − − EK digested sample − − − − − −

TABLE 20 SE probe Hybridization time (minutes) Digested sample 10 60 90120 180 900 SA digested sample − − − − − − SE digested sample − − − − −− PA digested sample − + + + + + EF digested sample − − − − − − EKdigested sample − − − − − −

TABLE 21 EF probe Hybridization time (minutes) Digested sample 10 60 90120 180 900 SA digested sample − − − − − − SE digested sample − − − − −− PA digested sample − − − − − − EF digested sample + + + + + + EKdigested sample − − − − − −

TABLE 22 EK probe Hybridization time (minutes) Digested sample 10 60 90120 180 900 SA digested sample − − − − − − SE digested sample − − − − −− PA digested sample − − − − − − EF digested sample − − − − − − EKdigested sample − + + + + +

Applications of these results obtained using the digested samples to thepresent invention could result in similar results. Therefore, theoptimal time period of hybridization in the identification of acausative microorganism of an infectious disease in the clinicalspecimen of the present invention was also set similarly.

Example 23 Influence of Surfactant Added to Hybridization Solution

The digested samples produced according to the process described inExample 17(1) [1] and [2] were used as specimens. When any of varioussurfactants (SDS, lauryl sarcosine, saponin, BRIJ35, Tween 20, TritonX-100) was added to the probe dilution solution followed byhybridization carried out according to Example 7, the detectionsensitivity was dramatically enhanced by adding 0.25% SDS. In addition,the detection sensitivity could be improved by lauryl sarcosine, BRIJ 35or Tween 20. The results are shown in Table 23 below.

TABLE 23 Surfactant Signal detection None added + SDS +++ Laurylsarcosine ++ Saponin + BRIJ 35 ++ Tween 20 ++ Triton X-100 +

Furthermore, as a consequence of using SDS at various concentrations, itwas revealed that preferable concentration was 1% or less, morepreferably 0.1% to 0.5%, and still more preferably 0.25%.

Applications of these results obtained using the digested samples to thepresent invention could result in similar results. Therefore, also inthe present invention, it is preferred that a surfactant, particularlySDS, is added at the step of in situ hybridization.

Example 24 Examination on Chain Length of Probe Used in Hybridization

Staphylococcus aureus probe (SA-24 (SEQ ID NO: 1)) and Pseudomonasaeruginosa probe (P2-2 (SEQ ID NO: 7)) were labelled with digoxigenin.

First, 1 μg of purified each type of the DNA probe was prepared to givethe total volume of 50 μg with 5 μl of 10×L.B. (0.5 mol/lTris-hydrochloric acid (pH 7.5), 50 mmol/l magnesium chloride, 5 μl of0.5 mg bovine serum albumin), 5 μl of 100 mmol/l dithiothreitol, each 1nmol of dNTPs (A, G, C), 0.5 nmol of digoxigenin-dUTP (Dig-dUTP), each0.5 nmol of dTTP, 3 μl of DNase (amount corresponding to 25 mU, 75 mUand 200 mU), 1 μl of 10 U/μl DNA polymerase and an appropriate amount ofsterile purified water. Digoxigenin labelling was performed at 15° C.for 2 hours. After the labelling, the mixture was boiled for 5 minutesto terminate the reaction. The reaction liquid after the termination wasinjected into a spin column (CENTRI-SEP COLUMNS CS901, PRINCETONSEPARATIONS, INC.), and centrifuged at 25° C. for 2 minutes (3,000×g) toremove free nucleotides. Then, concentration of the eluate was measuredby an absorbance meter. Electrophoresis was performed on a 3% agarosegel to confirm the size.

Next, DNA in the agarose gel was transferred to a nitrocellulosemembrane by Southern blotting method. Then, the membrane was immersed in2% blocking reagent (manufactured by Roche) for 30 minutes, andthereafter, alkaline phosphatase labelled anti-digoxigenin antibody inan amount of 1/5,000 was added thereto and the immersion was allowed for30 minutes. Next, the membrane was washed twice by shaking in 100 mmol/lTris-hydrochloric acid (pH 7.5) and 150 mmol/l sodium chloride for 10minutes. Thereafter, washing was executed by shaking in 100 mmol/lTris-hydrochloric acid (pH9.5) and 150 mmol/l sodium chloride for 10minutes. Then, color development was conducted by immersing in anNBT/BCIP solution.

Finally, the membrane was immersed in purified water to stop the colordevelopment, and dried. Consequently, as shown in FIG. 10 for (a) use ofthe SA probe and (b) use of the PA probe, respectively, it was indicatesthat in cases where cleavage was conducted using 25 mU of DNase (inFigure, lane 1) such that the chain length distributes the base lengthof predominantly about 350 to about 600, high labelling efficiency wasachieved. When thus resulting probe for detection was used in theprocess for detecting a causative microorganism of an infectious diseaseaccording to the present invention in which a digested sample or aclinical specimen from a patient suffering from an infectious diseasewas used to carry out hybridization, a signal could be detected withexcellent sensitivity. Therefore, it was reveled that chain length ofthe probe used in the hybridization may be the base length of about 350to about 600, and preferably the base length of about 350 to about 550.

Example 25 Examination on Probe Used in Hybridization

Escherichia coli digested samples produced according to the processdescribed in Example 17(1) [1] and [2] were used as specimens to examineon the probes for detection.

Probes for detection were prepared through labelling with digoxigenin asdescribed in Example 24 from EC-24 (SEQ ID NO: 11), EC-34 (SEQ ID NO:12) and EC-39 (SEQ ID NO: 13) such that they have the base length ofabout 350 to about 600, and used alone or in combination of those three,respectively. From thus obtained results, it was evident that the signalcould be detected more clearly resulting in elevated sensitivity for (d)the mixed probe MIX prepared by mixing the three ((a) EC-24, (b) EC-34and (c) EC-39), than for each (a) EC-24, (b) EC-34 or (c) EC-39 usedalone, as shown in FIG. 11.

INDUSTRIAL APPLICABILITY

Since the in situ hybridization according to the present method canoffer the stable signals within two hours or less, detection results canbe presented very rapidly. Obviously, such rapid detection demonstratesthe value of the present method in the rapid diagnosis of sepsis.

1. A method for detecting and/or identifying microorganisms causative ofinfectious diseases, comprising the steps of: (a) taking phagocytes froma clinical specimen and adjusting cell density of the phagocytes tobetween 5×10⁶ cells/ml and 1×10⁸ cells/ml; (b) immobilizing thephagocytes onto a solid support; (c) treating the phagocytes with lyticenzyme under the presence of protease inhibitor to increase phagocytecell membrane permeability and bare DNA in the phagocytes; (d) in situhybridizing the bared DNA with at least one DNA probe which canhybridize with DNA of at least one microorganism causative of aninfectious disease under conditions wherein the hybridization of the atleast one DNA probe is specific to the DNA of the at least onemicroorganism causative of an infectious disease; and (e) detectingand/or identifying the at least one microorganism causative of aninfectious disease by detecting hybridization the of at least one probeto the bared DNA.
 2. The method according to claim 1, wherein the lyticenzyme in step (c) is selected from the group consisting of Lysostaphinin a titer of from 1 Unit/ml to 1,000 Units/ml; Lysozyme in a titer offrom 1,000 Units/ml to 1,000,000 Units/ml; N-acetylmuramidase in a titerof from 10 Units/ml to 10,000 Units/ml; Zymolase in a titer of from 50Units/ml to 500 Units/ml, and a combination of any of the foregoing. 3.The method according to claim 1, wherein step (c) is performed under atemperature of about 26° C. to about 59° C. for about 15 to about 120minutes.
 4. The method according to claim 1, wherein the proteaseinhibitor in said step (c) is phenylmethylsulfonyl fluoride (PMSF). 5.The method according to claim 4, wherein the PMSF is employed at aconcentration of about 10 μmol/l to about 10 mmol/l.
 6. The methodaccording to claim 1, wherein the protease inhibitor in said step (c) isdissolved into dimethylsulfoxide (DMSO).
 7. The method according toclaim 6, wherein the DMSO is in a concentration of less than about 5% byvolume.
 8. The method according to claim 1, wherein step (d) isperformed at a temperature of about 25° C. to about 50° C. for about 30to about 900 minutes.
 9. The method according to claim 1, wherein thesolid support in step (b) is a slide coated with 3-aminopropyltriethoxysilane.
 10. The method according to claim 1, wherein step (e)comprises detecting signals resulting from hybridization, whereinpigment is employed to distinguish between said signals and cells. 11.The method according to claim 1, wherein the clinical specimen is blood.12. The method according to claim 1, wherein length of the at least oneDNA probe is from about 350 bases to about 600 bases.
 13. The methodaccording to claim 1, wherein concentration of the at least one DNAprobe is from about 0.1 ng/μl to about 2.2 ng/μl.
 14. The method ofclaim 1, wherein the in situ hybridizing of step (d) is performed in thepresence of a surfactant.
 15. The method according to claim 14, whereinthe surfactant is sodium dodecyl sulfate (SDS).
 16. A method fordiagnosing sepsis or bacteremia, comprising: (a) taking phagocytes froma clinical specimen and adjusting cell density of the phagocytes tobetween 5×10⁶ cells/ml and 1×10⁸ cells/ml; (b) immobilizing thephagocytes onto a solid support; (c) treating the phagocytes with lyticenzyme under the presence of protease inhibitor to increase phagocytecell membrane permeability and bare DNA in the phagocytes; (d) in situhybridizing the bared DNA with at least one DNA probe which canhybridize with DNA of at least one microorganism causative of sepsis orbacteremia under conditions wherein the hybridization of the at leastone DNA probe is specific to DNA of the at least one microorganismcausative of sepsis or bacteremia; and (e) diagnosing sepsis orbacteremia by detecting hybridization of at least one probe to the baredDNA.
 17. The method of claim 16, wherein the in situ hybridizing of step(d) is performed in the presence of a surfactant.